final colab notebook.doc

8/10/2019 Final Colab Notebook.doc

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Red Team Laboratory Notebook

Annie Aigster

Jonathon Goff

Sabrina Hannah

CHEM 5884Polymer Chemistry at the Biology Interface aboratory

S!ring "##$



Table of Contents

Size Exclusion Chromatography (SEC!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"#ntrinsic $iscosity!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! %&Thermal 'raimetric )nalysis!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!%*+ifferential Scanning Calorimetry (+SC!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!%,+ynamic -echanical )nalysis (+-)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!..Rheology!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!./'as Chromatography!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! "&Electron 0aramagnetic Resonance (E0R Spectroscopy!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! "*#n1Situ 2T#R Spectroscopy!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!*&)nalytical Sample 0reparation 3 -easuring c'04 5sing the 6xyScan System!!!!!!!!!!!!!!!!**6xygen Transmission Rate!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!7&+issoled 6xygen -easurements!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!7*)nalytical Sample Extraction!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!78)nalytical Sample Extraction!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!78Sensory Ealuation!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! /.)nimal Cell Culturing!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!/8

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Size Exclusion Chromatography (SEC

9asic 0rinciples:Si%e E&cl'sion Chromatogra!hy (SEC) is a se!aration techni*'e 'sef'l for characteri%ing the

molec'lar +eight an, molec'lar +eight ,istrib'tion of !olymers- In SEC. a !olymer sol'tion is!asse, thro'gh one or more ty!ically. se/eral. col'mns ha/ing ,iffering !ore si%es- 0he!olymer is se!arate, base, on its relati/e si%e in sol'tion 1no+n as the !olymer2s hy,ro,ynamic/ol'me- As the !olymer sol'tion flo+s thro'gh the col'mn the !olymer chains can mo/e in an,o't of the !ores ,e!en,ing on the si%e of the !ores an, the si%e of the !olymer chains- arger!olymers ,o not fit into as many !ores an, therefore !ass thro'gh the col'mn more *'ic1ly thansmaller !olymers- 0herefore. larger !olymers el'te first- SEC chromatograms !ro/i,e m'chinformation abo't !olymer molec'lar +eight an, chain branching- 0he n'mber a/erage. +eighta/erage. % a/erage. %3 a/erage. an, the !oly,is!ersity can all be calc'late, from the SEC,ata-

#nstrument 0rinciples: An SEC instr'ment contains se/eral col'mns arrange, in or,er of largest !ore si%e to smallest!ore si%e to form the col'mn ban1- 0he instr'ment can o!erate 'n,er organic or a*'eo'scon,itions. an, col'mns /ary accor,ingly- Col'mns can also be either !re!arati/e or analytical-In a ty!ical SEC e&!eriment the !olymer sol'tion is filtere,. then inecte, into the instr'ment- Aty!ical inection /ol'me is 5#6##( !er col'mn- As the !olymer sol'tion el'tes thro'gh thecol'mn. the !olymer is se!arate, base, on si%e- Common ,etectors for SEC instr'mentsincl',e refracti/e in,e& ,etectors. light scattering ,etectors. /iscometric ,etectors. an, masss!ectrosco!y ,etectors--ethod 0rinciples:SEC relies on generation of a calibration c'r/e- Sam!les of 1no+n an, narro+ (P7-)molec'lar +eight are r'n thro'gh the instr'ment an, the log of molec'lar +eight is !lotte,/erses el'tion /ol'me- As el'tion time9/ol'me ,e!en,s on hy,ro,ynamic ra,i's it is necessaryto 'se stan,ar,s +ith a str'ct're similar to the !olymer that one inten,s to characteri%e- 0he'n1no+n sam!le is r'n thro'gh the instr'ment at the same con,itions as the stan,ar,s- 0hesam!le !ea1 is ,i/i,e, into slices of el'tion /ol'me. :i an, area. niMi- 0he calibration c'r/e :ican then be 'se, to ,etermine the M; of each slice. Mi. an, s'bse*'ently the ,ifferingmolec'lar +eights an, !oly,is!ersity can be calc'late, accor,ing to the e*'ations incl',e,belo+- 0he instr'ment generally ma1es all calc'lations for the 'ser-Basic Sam!le <e*'irements=or a!!lication of SEC a sam!le m'st be sol'ble an, m'st be +ell ,issol/e, in the sol/ent !rior to inecting into the instr'ment- It is !referable to ha/e a ,il'te sol'tion so that the !olymer +ille&ist in its most rela&e, conformation res'lting in re,'ction of /iscosity effects an, non6re!ro,'cible retention- 0y!ical concentration is mg9m-

Typical +ata;Expected ResultsIn general res'lts from SEC analysis incl',e a chromatogram s'ch as those sho+n in the>Class <es'lts? section an, a re!ort incl',ing sam!le information. n'mber a/erage an, +eighta/erage molec'lar +eights an, !oly,is!ersity s'ch as that incl',e, in A!!en,i& -

Results from the Class Experiment Analysis of a Sample Using SEC In this laboratory e&!eriment SEC +as 'se to analy%e a !oly,is!erse !oly(ethylene o&i,e)sam!le re!orte, to be ##.### g9mol by the s'!!lier- 0he instr'ment 'se, is set '! to r'n

3



'n,er a*'eo's con,itions an, is connecte, to @ ,etectors a light scattering ,etector. a /iscosity,etector an, a refracti/e in,e& ,etector- In a,,ition to the ## PE sam!le. a!oly(methylmethacrylate) salt stan,ar, an, a "5.### g9mol PE stan,ar, +ere r'n to ens'rethat the instr'ment +as f'nctioning !ro!erly- 0he sam!les +ere !re!are, ahea, of time by <-Hy'c1- All sam!les +ere ,issol/e, at a concentration of mg9ml in +ater containing #-$MDaD@. #-M 0ris b'ffer an, "##!!m DaD@. a,'ste, to !HF-5 +ith acetic aci,- 0he light

scattering an, refracti/e in,e& traces for the PE stan,ar, sam!le are sho+n in =ig're . +hile=ig're " sho+s an o/erlay of the refracti/e in,e& traces for the PE stan,ar, an, the PEsam!le- 0he obtaine, molec'lar +eight /al'es for the analy%e, sam!le are sho+n in 0able -

2igure %! Chromatograms obtained for .7< 0E6 standard! The solid line indicatesthe light scattering trace =hile the dotted line indicates the refractie index trace!

2igure .! 6erlay of chromatograms for .7>&&&g;mol 0E6 standard and %&&>&&&g;mol0E6 sample! The .7>&&& g;mol 0E6 standard is sho=n in green (narro= -?distribution and the %&&>&&& g;mol 0E6 sample is sho=n in red (broad -?distribution! 9oth traces are from the refractie index detector! The black trace is thebaseline!

4



Table % -olecular =eight information forpolydisperse %&&>&&& g;mol 0E6 sample> asdetermined by SEC!

Mn 5$.## g9mol

M+ F.## g9mol

M% $$.8## g9mol

I M+9Mn -#8

Manual Calculation of Molecular Weight Using SEC Data As !art of the e&ercise. the refracti/e in,e& res!onse from !re/io'sly r'n e&!eriments +as 'se,to man'ally calc'late the molec'lar +eight of a ,ifferent PE sam!le- In or,er to !erformingthese calc'lations chromatograms of a series of stan,ar,s an, of the ,etermine, sam!le +eregenerate,- 0hese chromatograms an, the calibration c'r/e are sho+n belo+ in fig'res 46F-

2igure "! Chromatograms generated from refractie index trace of 0E6 standards!

5



2igure *! Calibration cure sho=ing linear model generated

from aboe standards! The generated e@uation relates -= toelution olume!

2igure 7! Chromatograms of the 0E6 sample generated from the refracie indexresponse! The corrected cure has the baseline subracted from the collected data!

0he calc'lations +ere ma,e by !erforming a n'merical integration of the sam!le c'r/e an,'sing the calibration c'r/e to ,etermine molec'lar +eight of each fraction- =rom the integration.the effecti/e mole /al'e. ni. an, the molec'lar +eight. Mi. of each fraction +ere obtaine,-0hese /al'es +ere then 'se, to calc'late the ,esire, molec'lar +eight /al'es from the

6



e*'ations sho+n in =ig're F- 0he obtaine, /al'es are re!orte, in 0able "-

2igure /! E@uations used to manually caluclate -n> -=> and -> =here ni is the molefraction of each piece in the numrical integration> -i is tne molecular =eight of eachfraction> and a is the -ark1Aou=ink constant =hich =as assumed to be &!/, for this excerci

Table .! -anually calculated molecular=eight alues for the 0E6 sample! Thesample =as reported to be .&&>&&& g;molby the supplier!

Mn @8.@# g9mol

M+ 84.8# g9mol

M/ 4F.F# g9mol

I M+9Mn 4-85

)pplications of the Techni@ue to 6xidation and the Chemistry19iology #nterface:SEC is a +i,ely 'se, techni*'e- It is an in/al'able techni*'e for both chemistry an, biologicallycentere, research- 0he ability to characteri%e the molec'lar +eight of !olymeric materials iscritical to s'ccessf'lly ,e/elo!ing 'sef'l materials- Molec'lar +eight characteri%ation enablesbetter ,e/elo!ment of !olymeric materials (both synthetic an, bio,eri/e,) for s'ch !'r!osesincl',ing foo, !ac1aging. ,r'g ,eli/ery. tiss'e scaffol,s. membranes. etc- As +ell. it is ,irectly

a!!licable to the st',y of o&i,ati/e !olymer ,egra,ation as it can be 'se, to e/al'ate, thee&tent of change in molec'lar +eight that a !olymer e&!eriences as a res'lt of o&i,ation- As+ell. o&i,ation is often in/ol/e, in the synthesis of !olymers an, in relation to this. SEC allo+sone to confirm s'ccessf'l synthesis of a !artic'lar molec'lar +eight !olymer- As a s!ecifice&am!le consi,er the +or1 ,one by ;olf. Macho an, e,erer- 0hese researchers 'se, SEC tomonitor the loss in molec'lar +eight e&!erience, by 'ltra6high molec'lar +eight !olyethylene,'e to o&i,ati/e ,egra,ation- 0his +or1 loo1e, at the effect of gamma ray sterili%ation. electorn

beam ra,iation crosslin1ing an, of incl',ing α6toco!herol in the materials- 0his material is 'se,

in me,ical ,e/ices an, therefore an 'n,erstan,ing of ho+ the material ,egra,es an, ho+ thetreatments being a!!lie, to it effect this ,egra,ation is critical- olarich et al 'se, SEC to

characteri%e α6!roteinase inhibitor (API) ,eri/e, from h'man !lasma an, 'se, as a

treatment for 6antitry!sin ,eficiency- 0he treatments a!!lie, to h'man !lasma to !re!are the API co'l, alter the glycosylation of the !rotein +hich +o'l, effect the !rotein fol,ing an,biological acti/ity- SEC analysis allo+e, the researchers to ,etermine the si%e of the APIsam!les an, the effect of treatments on them- 7elc'ratolo et al a!!lie, SEC to e/al'ate thee&ent of triglyceri,e !olymeri%ation in relation to o&i,ation of oli/e oils 'se, in the !re!aration offoccacia brea,- 0hese s!ecific e&am!les highlight the +i,e /ariety of a!!lications for thistechni*'e-

References

7



- ;olf. C- M- e,ere. - Accelerate, ageing e&!eriments +ith crosslin1e, an, con/entional'ltra6high molec'lar +eight !olyethylene (HM;6PE) stabili%e, +ith α 6toco!herol for total

oint arthro!lasty J Mater Sci: Mater Med. .&&/ $. @@@@4#-"- olarich. 7- 0'rece1. P-- ;eber. A- Mitterer. A- Graninger. M- Matthiessen. P- Dicolaes.

G-A-=- Altmann. =- Sch+ar%. H-P- Biochemical. molec'lar characteri%ation. an,

glyco!roteomic analyses of a6!roteinase inhibitor !ro,'cts 'se, for re!lacement thera!y- ransfusion! .&&/> 4F. 56$$-

@- 7elc'ratolo. 7- Gomes. 0- Para,iso. :-M- Dasti. <- am!arelli. =- E/ol'tion of theo&i,ation ,egree of e&tra /irgin oli/e oil 'se, for coo1ing ,ifferent 1in, of focaccia (italian flatbrea,)- "i#ista $taliana Delle Sostan%e &rasse- .&&/> 8@ (5) "@#6"@"-

)ppendix % 1 Light Scattering +ata for 0E6 from the SEC

)STR) 7!%!B Summary Report for )%&&<0E6%.!%B!.&&/D!af

Experiment name: CK7oc'ments an, SettingsKA,ministratorK7es1to!KAS0<A :

7A0AK"--"##FK A##PEL"--"##F-/afSample: ()0rocessing 6perator:Collection 6perator:

C6N2#'5R)T#6N

Light scattering instrument: mini7A;DCell type: 5Laser =aelength: F#-# nmCalibration constant: 8-$8@e6F 9(: cm)

R# #nstrument: Generic <I instr'mentSolent: #-$M DaD@. #-M 0ris. "##!!m DaD@. !HF (A,- 'sing glacial acetic aci,)

Refractie index: -@@42lo= rate: #-8## m9min

0R6CESS#N'

0rocessing time: ;e,nes,ay 7ecember "#. "##F #@#" PM Eastern Stan,ar, 0imeCollection time: 0'es,ay 7ecember . "##F # PM Eastern Stan,ar, 0ime+etectors used: " @Concentration detector: <I-ass results fitting: none (fit ,egree n9a)

Radius results fitting: none (fit degree: n/a)0eak %

0eak limits (min "4-F8 6 @-"4

dn;dc (mL;g #-@5). (mol mL;gF #-###-odel Nimm2it degree #nGected mass (g "-5F"5e64Calc! mass (g "-F5e64

RES5LTS

0eak %0olydispersity

8



-=;-n -#8($O) -z;-n -@"(8O)-olar mass moments (g;mol -n -5$e34(FO) -= -Fe34("O) -z -$$8e34(5O)

rms radius moments (nm Rn n9a R= @-8(#@O) Rz 4-@(4F8O)

9



#ntrinsic $iscosity

9asic 0rinciples:In a,,ition to the n'mber a/erage an, +eight a/erage molec'lar +eights. one can ,escribe the

si%e of a !olymer 'sing the /iscosity a/erage molec'lar +eight. M /- 0he e*'ations ,escribingthese /al'es are sho+n in =ig're - M/ lies bet+een Mn an, M+ b't is 's'ally closer to M+ thanMn- M/ can be e&!erimentally ,etermine, by meas'ring intrinsic /iscosity as ,escribe, inf'rther ,etail belo+-

2igure ,! E@uations describing number aerage> iscosity aerage> and =eight aerage

molecular =eight!

$nstrument 'rinciples:0he instr'ment 'se, to calc'late intrinsic /iscosity is a ca!illary/iscometer s'ch as a bbeloh,e /iscometer or Cannon6=ens1ety!e /iscometer- 0he instr'ment consists of a glass t'be an,b'lbs as sho+n in =ig're "- A sol'tion is intro,'ce, into the/iscometer then ,ra+n '! thro'gh the ca!illary !ortion- 0he timere*'ire, for the sol'tion to flo+ thro'gh a s!ecifie, ,istance ismeas're,- 0he /iscometer m'st be s's!en,e, in a +ater bath toens're constant tem!erat're con,itions ,'ring meas'rement-

Method 'rinciples:M/ can be e&!erimentally ,etermine, ,'e to its relation to the

intrinsic /iscosity (η) of the !olymer thro'gh the Mar16Ho'+in1

e*'ation LηM/a. +here an, a are constants- 0he constants are

s!ecific for a !artic'lar !olymer. in a !artic'lar sol/ent at a!artic'lar tem!erat're- 0he constant >? is a !ro!ortionalityconstant +hile >a? ,escribes the sha!e9stiffness of the !olymer insol'tion- 0he constant >a? generally ranges from #-5 to #-8 b't cantheoretically be any /al'e bet+een #. (the !olymer e&ists as har,s!heres) an, " (the !olymer e&ists as rigi, ro,s)-." 0heseconstants are e&!erimentally ,etermine, an, can be fo'n, in theliterat're- 0here are se/eral ,ifferent +ays to ,escribe /iscosity an,

these are s'mmari%e, in 0able -

Table "! $iscosity types and relationships> =here t is time and c is concentration in g;dL!Reproduced from class notes proided by $! Long!

Common Name 3 Symbol #50)C Name +efining E@uations

<elati/e :iscosity ηr :iscosity <atio ηr η9ηo ≈ t9to

S!ecific :iscosity 6 ηs! ηs! ηr 6(η6ηo) ≈ (t6to)9to

<e,'ce, :iscosity ηre, :iscosity D'mber ηre, ηs!9c

10



Inherent :iscosity ηinh ogarithmic :iscosity ηinh (lnηr )9c

Intrinsic :iscosity Lη imiting :iscosity D'mber Lη (ηs!9c)c→# L(lnηr )9cc→#

0he intrinsic /iscosity is also ,escribe, by the H'ggins an, the raemer e*'ations. both sho+nin =ig're @-

2igure 8! The Auggins and <raemer e@uations> =here kA and k< areconstants and c is concentration! Extrapolation of these e@uations to

zero concentration proides the intrinsic iscosity> D!

0he e&!eriment is !reforme, by intro,'cing sol/ent into the /iscometer an, meas'ring the timefor the sol/ent to flo+ thro'gh the in,icate, ,istance- S'bse*'ently. /arying 1no+nconcentrations of the sam!le are intro,'ce, into the t'be an, the effl'& times of these sam!lesare calc'late,- 0he time ,ata is 'se, to generate !lots of the H'ggins an, raemerrelationshi!s base, on the e*'ations !ro/i,e, in 0able - E&tra!olation of the generate, linesto %ero concentration !ro/i,es the intrinsic /iscosity- If ,ata is collecte, caref'lly thee&tra!olations sho'l, intersect at the same !oint-

9asic Sample Re@uirementsIn or,er to s'ccessf'lly a!!ly this techni*'e the sam!le m'st be in sol'tion an, the sol'tionconcentrations m'st be acc'rately 1no+n- As +ell. a!!ro!riate Mar16Ho'+in1 constants m'stbe ac*'ire, either from the literat're or e&!erimentally- 0he sam!les m'st be e*'ilibrate, to aconstant tem!erat're for time meas'rements to be acc'rate-

Typical +ata;Expected Results0he ,ata collecte, in this e&!eriment is the effl'& times for sol/ent an, /arying concentrations of !olymer sol'tions- 0his ,ata is 'se, to calc'late the s!ecific /iscosity an, the nat'ral log of therelati/e /iscosity +hich are then !lotte, /erses concentration- 0his generates a gra!h +ith t+olines- 0hese lines sho'l, ha/e the same y6interce!t. +hich is the intrinsic /iscosity- 0heintrinsic /iscosity is then 'se, to calc'late the /iscosity a/erage molec'lar +eight accor,ing tothe Mar16Ho'+in1 e*'ation- S'ch a !lot is sho+n in the ne&t section-

Results from the Class Experiment7'ring this class e&!eriment. intrinsic /iscosity +as 'se, to characteri%e the molec'lar +eight of a !oly(ethylene o&i,e) (PE) sam!le- 0he gra!h generate, from the ,ata is sho+n in =ig're 4-

11



2igure B! 'raph used to determine intrinsic iscosity of 0E6 sample!

0he intrinsic /iscosity +as fo'n, to be -@ from the H'ggins e*'ation an, -@5 from theraemer e*'ation- 0hese /al'es +ere 'se, to calc'late M/ 'sing the Mar16Ho'+in1 constantsa #-F$ an, 4- & # 6@ m9g obtaine, from the literat're-@ 0he calc'late, M/ /al'es +erea/erage, to obtain an M/ of ".F## g9mol for the PE sam!le-

)pplications of the Techni@ue to 6xidation and the Chemistry19iology #nterface0he techni*'e of meas'ring intrinsic /iscosity to characteri%e molec'lar +eight is +i,ely 'se, inthe recent literat're- ' an, ;ang4 'se, this techni*'e to st',y the o&i,ati/e ,egra,ation of!olyethylene films- 0hese researchers st',ie, the effect of incl',ing =e@3 an, starch in the!olyethylene matri& +ith the aim of enhancing ,egra,ation ,'ring com!osting- Meas'rement of intrinsic /iscosity +as also 'se, by Botelhoa et al-5 to st',y o&i,ati/e ,egra,ation of a !olymer

in the conte&t of enhancing ,egra,ation- Botelhoa et al- st',ie, the effect of co/alentmo,ification of styrene on the ,egra,ation of !olystyrene- 0hese +or1s relate to both o&i,ation.as the researchers +ere loo1ing at o&i,ati/e ,egra,ation. an, also to the chemistry6biologyinterface as the +or1 is aime, at i,entifying +ays to ma1e more en/ironmentally frien,ly !lasticmaterials- Partic'larly the +or1 of ' an, ;ang in/ol/es research at the interface of these t+o,isci!lines in that their +or1 consi,ers the effect of the bio!olymer starch- ee. Bo'ha,ir an,MooneyF also a!!lie, this techni*'e to the e/al'ation of a bio!olymer- In this case theresearchers +ere in/estigating the effect of o&i,ation on the chain stiffness of !olyg'l'ronate. a!olysacchari,e- 0hese researchers 'se, meas'rement of intrinsic /iscosity in con'nction +ithsi%e e&cl'sion chromatogra!hy to e/al'ate chain stiffness as reflecte, in the Mar16Ho'+in1constant >a-?

References- Painter. P-C- Coleman. M- M- (undamentals of 'olymer Science. 0echnomic ancaster.

$-"- +++-!olymerchemistryhy!erte&t-com9mar1Qho'+in1-htm@- Pro/i,e, in class notes. /al'es are from 4th e,ition of the Polymer Han,boo14- '. J-G- ;ang. R- 7- St',ies of enhancing effect of =e@3 on ,egra,ation of blen,s of starch

an, !olyethylene- Acta 'olymerica Sinica .&&%> . 6#4-

12



5- Botelhoa. G- 'eirosa. A- Macha,ob. A- =rangiosac. P- =erreira. J- Enhancement of thethermoo&i,ati/e ,egra,ability of !olystyrene by chemical mo,ification- 'olymerDegradation and Sta)ility .&&*> 8F. 4@64$-

F- ee. - - Bo'ha,ir. . H- Mooney. 7-J- E/al'ation of Chain Stiffness of Partially &i,i%e,Polyg'l'ronate- *iomacromolecules . .&&.> @ (F). "6@4-

13



Thermal 'raimetric )nalysis

9asic 0rinciple0hermogra/imetric analysis (0GA) meas'res +eight changes in materials as a f'nction of

tem!erat're 'n,er a controlle, atmos!here (nitrogen)- 0he basic !rinci!le of 0GA is tomeas're the materialTs thermal stability an, com!osition- 0hermal gra/imetric analysis is's'ally !erforme, before 7SC analysis of ne+ materials" to ,etermine the tem!erat're at +hicha sam!le ,egra,es-@ no+ing the ,ecom!osition tem!erat're of a sam!le allo+s for ana!!ro!riate 7SC !rogram to be ,e/elo!e, for that !artic'lar sam!le-

0he metho, an, instr'ment s!ecifications 'se, in this e&!eriment incl',e, 'sing a !latin'msam!le !an ,'e to its resistance to high tem!erat'res- Al'min'm !ans ,egra,e at F## UC+hereas !latin'm !ans can s'stain tem!erat'res '! to ### UC- 0he sam!le si%e 'se, range,from abo't @65 mg 'n,er D" gas F# m9min- 0he !rogram con,itions +ere to start Vambient tem!erat're. ram! V "# WC9min to F## WC-

Sam!le ,escri!tion n!rocesse, H7PE <esin0reatment S'bmerge, in "-5O Cloro&X Bleach. " ,ays. $# WC 9asic Sample Re@uirements0he sam!le si%e 'se, is bet+een @65 mg an, is !lace, in the !latin'm !an- 0he sam!le is c'tfrom a thin layer of resin an, it +as not cr'she,-

Typical +ata or Expected Results and Results from the Class Experiment

2igure %! +egradation Temperatures of Control and Treated A+0E Resins!This figure sho=s that the treated A+0E resin degraded at a lo=er temperaturecompared to the control A+0E resin!

14



2igure .! ?eight Changes of Control A+0E Resin!

0his fig're sho+s the +eight changes in the control H7PE resin- At the ,egra,ationtem!erat're. the +eight of the sam!le ra!i,ly ,ecrease,-

)pplication of the Techni@ue to 6xidation and Chemistry19iology #nterface0hermogra/imetric analysis is a commonly 'se, research techni*'e to characteri%e. ,etermine,egra,ation tem!erat'res. an, absorbe, moist're content in !olymeric materials-0sai an, o@ meas're, thermal !ro!erties of a ne+ hybri, film 'sing 0GA- 0he film teste, +ere!oly(imi,e silo&ane) (PIS) an, PIS90antal'm o&i,e (0a&) hybri, films- 0he res'lts sho+e, that

the hybri, film ,ecom!ose, at a lo+er tem!erat're com!are, to the PIS films- 0he thermal,egra,ation of the hybri, film +as attrib'te, to metallic resi,'e +hich can o&i,ati/ely ,egra,ethe !olyimi,e !olymer film-@

Another st',y sho+e, that chemical mo,ification of chitosan +ith 46/inyl!yri,ine (:P) res'lte,in increase, thermal stability of the !olymers-4

Altho'gh charateri%ation of !olymers is a big a!!lication of 0GA. this techni*'e can also be'se, in a /ariety of nat'ral biomolec'les (s'ch as cyclo,e&trins an, chitosan) an, theirf'nctional !ro!erties altere, by chemical an, or biological mo,ifications- Chan et al-5 'se, 0GAan, other thermal an, mechanical techni*'es to characteri%e cyclo,e&trin !olymers an,e/al'ate the ,egra,ation tem!erat'res of /ario's !olymers-

References!

- htt!99+++-tainstr'ments-com"- Class Dotes 6 0im ong. "##F-@- 0sai. MH o. CJ- In sit' =ormation. S'rface Characteristics. an, Interfacial A,hesion of

!oly(imi,e silo&ane)9tantal'm o&i,e Hybri, =ilms- Surf. Coat. ech. .&&/. "#. 4@F$64@$-4- El1holy. SS halil. 7 Elsabee. MN- Homogeneo's an, Heterogeneo's Grafting of 46

:inyl!yri,ine onto Chitosan- J. Appl. 'olym. Sci. .&&/. . @@#86@@$-5- Chan. SC 'o. S; She. HS in. HM ee. H= Chang. =C- S'!ramolec'lar Aggregations

thro'gh the Incl'sion Com!le&ation of Cyclo,e&trins an, Polymers +ith B'l1y en, Gro'!s-J. 'olym. Sci. 'art A: 'olym Chem. .&&,. 45. "56@5-

15



16



+ifferential Scanning Calorimetry (+SC

9asic 0rinciples7ifferential scanning calorimetry (7SC) is a thermal analysis techni*'e commonly 'se, to

meas're en,othermic (heat flo+ into sam!le) an, e&othermic (heat flo+ o't sam!le) transitionsas a f'nction of tem!erat're- 0he transitions meas're, +ith a 7SC instr'ment incl',e glasstransition (0g). crystalli%ation. melting. c'ring 1inetics. onset of o&i,ation. an, heat ca!acity-0he ,ifference in the amo'nt of heat flo+ bet+een a sam!le an, a reference ('s'ally an em!ty!an) is meas're, as a f'nction of time an, tem!erat're- Both the sam!le an, reference !ansare maintaine, at nearly the same tem!erat're- 0h's. the basic !rinci!le of 7SC is to meas'rethe ,ifference in heat flo+ bet+een a sam!le an, a reference at a ,etermine, rate- ;hen asam!le 'n,ergoes !hase transitions. more or less heat flo+ is nee,e, ,e!en,ing on +hetherthe transitions are en,othermic or e&othermic- ;hen a soli, melts. more energy is re*'ire, an,the !rocess is en,othermic- ;hen a sam!le 'n,ergoes crystalli%ation (e&othermic). less heat isre*'ire, by the sam!le- 0his meas'res the amo'nt of heat absorbe, or release, by the sam!lerelati/e to the reference ,'ring !hase transitions-

0he 7SC instr'ment consists of a cell that contains a sam!le !an an, a reference !an- 0he!ans are 's'ally ma,e of al'min'm- 0he !ans are crim!e, an, the heat flo+ ,ifferencebet+een the t+o !ans is monitore, 's'ally by increasing the tem!erat're at a s!ecific rate('s'ally # UC9min)- 7'ring the r'n. the ,ifference in heat flo+ bet+een the sam!le an,reference is recor,e, in a com!'ter- 0he r'n lasts for abo't ho'r ,e!en,ing on the 7SCo!erating con,itions- ;hen !hase changes are obser/e,. an '!+ar, !ea1 +ill sho+e&othermic changes (crystalli%ation. cross6lin1ing. o&i,ation) an, a ,o+n+ar, !ea1 +ill sho+en,othermic changes (0g. melting) on the 7SC gra!h-

In o'r e&!eriment. high ,ensity !olyethylene (H7PE) resins e&!ose, to YF5.### !!m freechlorine (chloro&X bleach) at !H4-#. $#UC for " ,ays +ere e/al'ate, 'sing 7SC- 0he first!art of the e&!eriment 'se, the heat9cool9heat metho, to ,etermine the 0c an, 0m. an,calc'lations of the crystallinity of the treate, H7PE !olymer- 0he secon, !art of the e&!erimente/al'ate, the o&i,ati/e in,'ction time (I0) of the chlorine treate, H7PE resin com!are, to the'ntreate, H7PE resin-"

9asic Sample Re@uirements0he sam!le si%e 'se, in 7SC thermal analysis /aries from Y 5mg to Y"# mg ,e!en,ing on the!an si%e an, sam!le- Small sam!le si%e (Y@ to 5 mg) is 'se, for metals. +hereas #65 mgsam!le si%e is 'se, for !olymers- s'ally a # mg sam!le is 'se,- In o'r e&!eriments Y 5 mgresin sam!les +ere 'se,- 0he sam!le !re!aration in/ol/e, 'sing c'tting a thin !iece of theH7PE treate, resin that +as not cr'she,- ;hen a sam!le contains /olatile com!o'n,s.!inholes on the li, of the !an are ma,e to allo+ e/a!oration an, !re/ent e&!losion of the !anthat co'l, !otentially ,amage the e*'i!ment an, the o!erator-

Typical +ata or Expected Results A ty!ical 7SC !hase transition com!osite thermogram is as sho+n in =ig're -

17



2igure %! Schematic +SC cure of common phase transitions!(http:;;===!ans=ers!com;topic;differential1scanning1calorimetry "

Results from the Class ExperimentPhase transitions of the H7PE resin treate, +ith chlorine- Al'min'm !ans +ere 'se, for thesam!le an, reference (em!ty !an)- 0he !ans +ere crim!e, +ith li,s- 0he 7SC con,itions+ere start V @# UC. ram! V "# UC9min to "## UC. cool to "# UC V UC9min

a) 0hermal transition 6 0m

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7SC=ile C---K"#7ecoFPolyabKH7PE treate,6-##

<'n 7ate "##F6"6"# 4@Instr'ment 7SC ### :-# B'il, "$5

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2igure .! Thermal transition temperature 1 Tm of A+0E resin treated =ith chlorine!

18

Exo



0m @"-8 UC

Area 'n,er the melting c'r/e 8F-4 J9g0heoretical /al'e for ##O crystalline H7PE " J9g

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7SC=ile C---K"#7ecoFPolyabKH7PE treate,6-##

<'n 7ate "##F6"6"# 4@Instr'ment 7SC ### :-# B'il, "$5


2igure "! Thermal transition temperature 1 Tc of A+0E resin treated =ith chlorine!

19



c) &i,ation in,'ction time (I0) of treate, an, 'ntreate, H7PE resins- Con,itions al'min'msam!le !an no li,s. ram! "# [C9min to "## [C 'n,er D"- Hol, 5 min'tes-S+itch to "

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2igure *! 6xidation induction time (6#T of A+0E resin treated =ith chlorine and untreatedA+0E resin!

&i,ation in,'ction time of treate, resin F-@ min

&i,ation in,'ction time of 'ntreate, resin @@-$ min

0he I0 of the treate, is shorter in,icating that chlorine treatment res'lte, in increase,o&i,ation of the !olymer com!are, to the control-

)pplication of the Techni@ue to 6xidation and the Chemistry19iology #nterface7ifferential scanning calorimetry is a commonly 'se, techni*'e to ,etermine !hase transitionsof materials by monitoring the ,ifference in heat flo+ (in an o't) of the sam!le relati/e to thereference at a s!ecific rate- 0his techni*'e is +i,ely 'se, to characteri%e synthetic an, nat'ral!olymers. foo,s. biological sam!les. !harmace'ticals an, other chemical com!o'n,s- Ina,,ition. cross6lin1ing. onset of o&i,ation. an, heat ca!acity can be meas're, from 7SCthermograms-

0his techni*'e can be 'se, to st',y the o&i,ati/e stability of li!i, systems s'ch as lecithin an,linoleic aci,-4 In a,,ition 7SC can be 'se, to characteri%e the thermal !ro!erties s'ch as 0g ofbio,egra,able !olymers s'ch as !oly/inyl alcohol9lactic aci, !olymeric films that can be 'se,for me,ical fiel, an,9or foo, !ac1aging a!!lications-5 Moreo/er. 7SC can be 'se, to assess the thermal stability of antio&i,ants in li!i, systems s'chas !'re oils-F

References

20

Treated

Untreated



- Class Dotes 6 7r- 0im ong. "##F-"- aboratory Han,o't. "##$-@- htt!99+++-ans+ers-com9to!ic9,ifferential6scanning6calorimetry-4- l1o+s1i. M M'siali1. M it+inien1o. G- se of 7ifferential Scanning Calorimetry to St',y

i!i, &i,ation- - &i,ati/e Stability of ecithin an, inoleic Aci,- J. Agric. (ood Chem..&&7> 5@. #$@6#$$-

5- Se,lari1. : Saha. D 'rit1a. I Saha. P- Pre!aration an, Characteri%ation of Poly (/inylalcohol)9actic Aci, Com!o'n,e, Polymeric =ilms- $nternational J 'olym. Anal. Charact..&&/> . "5@6"$#-

F- Gi'ffri,a. = 7estaillats. = Herrero Hegart. M H'g. B Golay. PA S1ibste,. H 7ionisi. =- Acti/ity an, 0hermal Stability of Antio&i,ants by 7ifferential Scanning Calorimetry an,Electron S!in <esonance S!ectrosco!y-

21



+ynamic -echanical )nalysis (+-)

9asic 0rinciples$nstrument 'rinciples

7ynamic mechanical analysis (7MA) is a characteri%ation techni*'e that is 'se, to st',y the/iscoelastic !ro!erties of a material- Stiffness an, ,am!ing are the t+o material !ro!ertiesmeas're, +ith this instr'ment- Stiffness relates to the mechanical !ro!erties of a material. an,is meas're, by ,etermining sam!le ,is!lacement from an oscillating force- Mo,'l's can thenbe calc'late, from the stiffness of a material- 7am!ing. +hich is !ro!ortional to the energy amaterial can store. is meas're, from the time lag in sam!le ,is!lacement in relation to the

a!!lie, oscillating force (=ig're )-6" 0his material !ro!erty is e&!resse, as 0an δ- 7MA

generates storage mo,'l's an, 0an δ ,ata as a f'nction of tem!erat're- Generating a !lot of

this ,ata allo+s for ,etermination of the glass transition tem!erat're (0g) an, /ario'sengineering *'antities (i-e- fail're mo,es. fre*'ency ,e!en,ence. an, o'ng2s mo,'l's) of amaterial-6@

2igure %! +etermination of the damping factor> Tan %

0he o!eration of a 7MA instr'ment first in/ol/es a force being a!!lie, to a sam!le by a ,ri/eshaft connecte, to a motor- 0he a!!lie, oscillatory force is sin'soi,al in nat're +ith a fi&e,fre*'ency- n,er stress. the sam!le ,eforms. an, a sensor meas'res the ,is!lacement-6@ =ig're " sho+s a schematic of a 7MA instr'ment-"

2igure .! ) schematic of a +-) sho=ing the motor> L$+T positional sensor>and sample setup.

As state, earlier. ,'ring the meas'rements of mo,'l's an, 0an δ. tem!erat're can be change,.

allo+ing for the generation of tem!erat're ,e!en,ent !lots of stiffness an, ,am!ing- =or thisreason. 7MA is sometimes referre, to as ,ynamic mechanical thermal analysis (7M0A)-" AS0Mmetho,s for 7MA ,escribe the /ario's ty!es of heating ro'tines em!loye, +ith this instr'ment-

22



0hese ro'tines incl',e ste!+ise heating (Y5#C e/ery fe+ min'tes) an, contin'o's heating (Y6"#C !er min'te)-@ It is '! to the 'ser to ,eci,e +hich ro'tine is a!!ro!riate for a s!ecificsam!le-

Method 'rinciples0he metho, !rinci!les that corres!on, +ith 7MA rely '!on the /iscoeleastic beha/ior of

materials- A brief o/er/ie+ of these beha/iors +as ,escribe, in the !re/io's section- Materialsthat e&hibit /iscoelastic beha/ior can be fo'n, in t+o contrasting states- 0hese materials,is!lay glassy beha/ior (high mo,'l's /al'e) at lo+er tem!erat'res an, r'bbery beha/ior (lo+mo,'l's /al'e) at higher tem!erat'res- 0he tem!erat're at +hich the /iscoelastic materials+itches bet+een these t+o ,istinct states is referre, to as the 0g-

@ 7MA instr'mentation isca!able of meas'ring the 0g of a material. as +ell as the time ,e!en,ent feat'res of thetransistion- ;hile other techni*'es are ca!able of meas'ring the transition (i-e- 7SC). 7MA isthe most sensiti/e techni*'e for the meas'rement of this transition an, other rela&ationmeas'rements-.@

9asic Sample Re@uirements0he sam!le re*'irements for this techni*'e are not /ery restricti/e- Sam!les can be mo'nte,

in se/eral ,ifferent geometries +hen 'sing 7MA- 7eci,ing +hich geometry to 'se ,e!en,s onsam!le characteristics an, the ,esire, meas'rements- =ig're @ sho+s si& common sam!legeometries 'se, +ith this techni*'e-"

2igure "! Six different sample geometries employed in +-).

Typical +ata or Expected Results

0y!ical ,ata obtaine, from 7MA is a !lot of storage mo,'l's an, 0an δ as a f'nction of

tem!erat're- Glass transition tem!erat're. sam!le stiffness. rela&ation meas'rements. fail'remo,es. an, time6,e!en,ent transitions. to name a fe+. can be ,etermine, for a /iscoelasticmaterial- =ig're 4 sho+s a 7MA !lot of !oly(methyl methacrylate) (PMMA)-"

23



2igure *! +-) plot of 0--).

Results from the Class Experiment0+o H7PE resins ('ntreate, an, treate,. co'rtesy of An,re+ ;helton) +ere r'n on 7MA ,'ring

the class e&!eriment- 0he treate, H7PE sam!le +as treate, +ith bleach sol'tion (containingYF5.### !!m free chlori,e) for three +ee1s at $##C- <es'lts sho+n in !lot belo+

6"-@GWC

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ni/ersal :@-GA 0A Instr'ments

)pplication of the Techni@ue to 6xidation and the Chemistry19iology #nterfaceiterat're re/eals many recent m'lti,isci!linary a!!lications of ,ynamic mechanical analysis-Hoso,a et al- 'se, 7MA to e/al'ate the mechanical !ro!erties of biome,ical sha!e memoryalloys- 0his !o+erf'l techni*'e enable, the a'thors to sim'ltaneo'sly ,etermine the stress.strain. storage9loss mo,'l's. internal friction. an, tem!erat're of the alloys-4 ther research.!erforme, by Sil/a et al-. also 'tili%e, 7MA to characteri%e the mechanical !ro!erties of sha!e6

24



memory !oly'rethanes 'se, for their ability to interact +ith h'man fibroblasts an, !latelets-5 ne final e&am!le of a recent a!!lication of 7MA at the chemistry6biology interface in/ol/es thest',y of chitosan membranes-F 0he mechanical !ro!erties of these membranes +eremeas're, +ith 7MA in sim'late, !hysiological con,itions- Biome,ical materials benefit from7MA characteri%ation by hel!ing the researchers to ,esign ,'rable materials for biologicalsystems-

References- 0a1emore. M- SPEAD0EC- ($8) +,! 6@"-"- 0riton 0echnology- ;hat is 7ynamic Mechanical Analysis. 7MA\ nite, ing,om 0riton.

"##@-@- >7ynamic Mechanical Analysis-? ;i1i!ei,a- "##F-

htt!99en-+i1i!e,ia-org9+i1i97ynamicQmechanicalQanalysis-4- Hoso,a. Hi,e1i ='1'i. 's'1e ;a1ashima. eni Miya%a1i. Sh'ichi- ransactions of the

Materials "esearch Society of Japan ("##@). +- (@). 56F#"-5- =are Sil/ia :alt'lina :i/iana Petrini Paola Alessan,rini E,oar,o Pietrocola Giam!iero

0an%i M Cristina S!e%iale Pietro :isai i/ia- Journal of )iomedical materials research. 'art A ("##5). /(). 6-

F- Sil/a < M Sil/a G A Co'tinho P Mano J = <eis < Journal of materials science.Materials in medicine ("##4). 01 (#). #56"-

25



Rheology


<heology is a fiel, that in/ol/es the st',y of the flo+ of materials- 0his st',y is ty!ically!erforme, +ith the material 'n,er a!!lie, force- Instr'mentation in this ,isci!line allo+s for the,etermination of elasticity (soli,s). /iscosity (li*'i,s). an, fl'i, mechanics (De+tonian /s- Don6De+tonian fl'i,s)-64 All of the meas're, !ro!erties in rheology are a f'nction of the shear ratea!!lie, to the material- 0he e&!erimental techni*'es 'se, to ,etermine the rheological!ro!erties of a material are referre, to as rheometry- <heometry has a!!lications in many,i/erse fiel,s. s'ch as engineering. !hysics. !hysiology. an, geology-"64

<heometry of fl'i,s re*'ires the generation of shear flo+- A sim!le metho, to generate shearflo+ in/ol/es !arallel !lates- =ig're sho+s a schematic for a !arallel !late rheometer- In thisset'!. a fl'i, lies bet+een t+o !arallel !lates- 0he bottom !late is fi&e,. +hereas the to! !lateis free to mo/e- 0he to! !late mo/es across the fl'i, at a constant rate. creating a force- 0heshear rate a!!lie, to the fl'i, can be calc'late, by ,i/i,ing the mo/ement rate of the to! !lateby the se!aration bet+een the t+o !lates-"6@ 0he rheometry instr'mentation meas'res the/iscosity of the fl'i, at each a!!lie, shear rate- ther set'!s may be 'se, to create shear flo+in rheometry. incl',ing ca!illary t'bes an, cone6an,6!late ,esigns-6@

<heometers that contain !arallel !late an, cone6an,6!late set'!s are ca!able of !erformingoscillatory shear flo+ e&!eriments- 0his in/ol/es the to! !late9cone a!!lying a sin'soi,al force(stress) to the sam!le- =rom the meas're, strain of the sam!le. storage mo,'l's (G2 LPa) an,/iscosity (Pa s) can be calc'late,- Another ty!e of stress. referre, to as normal stresses. canbe generate, in rheometry 'sing a cone6an,6!late set'!- In the e&!eriment !erforme, in thislab. a !arallel !late oscillatory set'! +as 'se,-

2igure %! 0arallel plate setup to generate shear flo= of a fluid %

Method 'rinciples=l'i,s can be ,escribe, as De+tonian or Don6De+tonian- <heometry is 'se, to ,etermine theclassification of a fl'i, base, on ho+ it beha/es +hen shear stress is a!!lie,- De+tonian fl'i,s,is!lay a linear stress9stress c'r/e- 0he /iscosities of these ty!es of fl'i,s are constant.regar,less of the shear rate- Don6ne+tonian fl'i,s ,is!lay a non6linear stress9strain c'r/e. an,their /iscosities are ,e!en,ent '!on shear rate- =ig're " sho+s the stress9shear rate c'r/es for the t+o ty!es of fl'i,s-4

26



2igure .! Stress;Strain Cures for Ne=tonian and Non1Ne=tonian 2luid*

9asic Sample Re@uirements0he sam!le re*'irements for rheometry techni*'es ,e!en, on the set'! 'se,- 0he /ario'sty!es of set'!s incl',e !arallel !late (sho+n abo/e). ca!illary t'be. cone6an,6!late. an,Co'ette flo+- =ig're @ sho+s the schematics of these ,ifferent set'!s-

2igure "! Schematic of different deices used in rheometry%

27



Typical +ata or Expected Results=ig're 4 sho+s a ty!ical !lot generate, 'sing rheometry techni*'es of /ario's !olymersol'tions- 0he !lot sho+s /iscosity (Pa s) as a f'nction of shear rate-

2igure *! 0lot of iscosity s! shear rate%

Results from the Class ExperimentCirc'lar sam!les +ere !re!are, to fit the rheometer 'se, in these e&!eriments- An oscillatoryrheological techni*'e +as !erforme,- 0he t+o con,itions obser/e, in the e&!eriment +ere5##C +ith hollo+ circles an, ##C +ith soli, circles- =ig're 5 sho+s the !lot of the res'ltsfrom the class e&!eriment-

28



#-#4### #-4### 4-### 4#-## 4##-#fre*'ency (H%)

4###

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2igure 7! Results from class experiment

)pplication of the Techni@ue to 6xidation and the Chemistry19iology #nterface<ecent a!!lications of rheology in m'lti,isci!linary research incl',e ) se of rheology in thest',y of the role of =6actin an, myosin in e!ithelial cell rheology- 0he researchers concl',e,that both =6actin an, myosin ,o not affect the rheological !ro!erties of e!ithelial cells- 5 ") St',yof the rheological characteristics of o&i,i%e, !otato starch- &i,i%e, !otato starch +ith lo+,egrees of s'bstit'tion form gels- 0he scientists fo'n, that the ,egree of o&i,ation ha, a largeeffect on these rheological !ro!erties-F

References

- Hinch. E- J- ect're Intro,'ction to <heology- "##4. 6-"- ><heology-? ;i1i!e,ia- "##F- htt!99en-+i1i!e,ia-org9+i1i9<heology-@- ><heometry-? ;i1i!e,ia- "##F- htt!99en-+i1i!e,ia-org9+i1i9<heometry-4- >Don6De+tonian =l'i,-? ;i1i!e,ia- "##F- htt!99en-+i1i!e,ia-org9+i1i9Don6De+tonianQfl'i,-5- :an Citters. athleen M- Hoffman. Brenton 7- Massiera. Gla,ys Croc1er. John C-

*iophysical Journal . .&&/. 20(#). @4F6@5F-F- Sa,o+s1a. Ja,+iga ;ron1o+s1a. Malgor%ata e+an,o+ic%. Gra%yna Soral6Smietana.

Maria. 'olish Journal of (ood and 3utrition Sciences .&&/. 01 (@). @6@8-

29



'as Chromatography


Gas chromatogra!hy (GC) is a chromatogra!hic techni*'e that 'ses a carrier gas mobile!hase. +hich is !asse, thro'gh a col'mn that is !ac1e, +ith a li*'i, or !olymeric stationary!hase- An inert gas (i-e- hy,rogen. heli'm. or nitrogen) is 'se, as the mobile !hase- In thecase of this lab e&!eriment. heli'm +as em!loye, as the carrier gas- 0he gas carries thesam!le thro'gh the col'mn. +here the analytes that com!rise the sam!le are se!arate, base,on !olarity an, boiling !oint- Col'mns 'se, in GC are ,i/i,e, into t+o ,ifferent classifications.ca!illary an, !ac1e,-6"

0he e&!erimental set'! of a gas chromatogra!h consists of se/eral systems connecte,together- =irst. there is the carrier gas tan1. +hich is connecte, to a flo+ control system thatallo+s for the release of the inert gas at a constant flo+ rate- De&t. there is the sam!le inlet.+here the sam!le is intro,'ce, to the carrier gas mobile !hase- A ty!ical inlet system 'tili%esthe hot /a!ori%ing inection metho,. +here the sam!le is heate,. /a!ori%e,. an, mi&e, +ith thecarrier gas- Accor,ing to the GC !resentation in class. s!lit inection is the easiest inlettechni*'e 'se, in e&!eriments- After the /a!ori%e, sam!le is mi&e, +ith the carrier gas. they!ass thro'gh the heate, col'mn- 0he stationary !hases in to,ay2s GC instr'ments arecom!ose, of either silo&anes or !olyethylene glycol-6" ;hen a sam!le is non!olar. it isse!arate, by its boiling !oint- Polar < gro'! can be a,,e, to the stationary !hase. changingthe !olarity of the col'mn. an, hence changing the se!aration of the sam!le- After the sam!leis se!arate, in the col'mn. it then contin'es to the ,etector an, recor,er- 0here are manyty!es of ,etectors 'se, in GC. b't flame ioni%ation ,etectors are by far the most common-=ig're sho+s a sim!le schematic of a GC-

2igure %! Schematic of a gas chromatograph%

Method 'rinciplesChromatogra!hy is a techni*'e that aims to se!arate the in,i/i,'al com!onents in a sam!le-0his se!aration techni*'e hel!s a 'ser to ,etermine the chemical i,entities an, !'rity ofmaterials- ther ty!es of chromatogra!hy incl',e thin layer chromatogra!hy. col'mn

30



chromatogra!hy. an, si%e e&cl'sion chromatogra!hy- Gas chromatogra!hy +as ,e/elo!e, inthe 5#2s. an, has been 'se, e/er since as an analytical tool in a +i,e /ariety of ,isci!lines-6"

In chromatogra!hy. the se!aration of com!onents occ'rs from the interaction of the sam!lebet+een a mobile !hase an, a stationary !hase- 0he stationary !hase in GC is ty!ically a li*'i,that is bon,e, to a soli, s'!!ort insi,e the col'mn-6" Analytes are retaine,9se!arate, base, on

the amo'nt of !hysical a,sor!tion to the stationary !hase that occ'rs- As state, earlier. the!olarity of col'mns can be a,'ste,. allo+ing for a 'ser to fin, an o!timal stationary !hase for as!ecific sam!le- <etention time is ,efine, as the amo'nt of time bet+een inection an,,etection of a chemical- 0he retention time ,e!en,s '!on the mobile !hase flo+ rate. an, theamo'nt of time the chemical s!en,s in the stationary !hase an, the mobile !hase- Col'mn an,flo+ rate sho'l, be a,'ste, to obtain a s!ectr'm that !ro/i,es goo, resol'tion (se!aration) ofthe analytes in a sam!le- =ig're " sho+s a !lot ill'strating the ,ifferences in retention timebet+een a sol'te an, an analyte in a sam!le-

2igure .! +ifference in retention time of a solute and an analyte %

9asic Sample Re@uirementsSmall sam!le si%es are 'se, in GC. an, microsyringes are ty!ically 'se, to inect the sam!le-

µ of sam!le sol'tion is a,e*'ate for most col'mns 'se, in GC- Slo+ inection times sho'l, be

a/oi,e, in GC. as this lea,s to a loss of resol'tion in the corres!on,ing s!ectr'm- =inally. toens're instant e/a!oration of the sam!le. the tem!erat're of the inector !ort nee,s to be high-

As a general r'le. the tem!erat're of the inector !ort sho'l, be Y5##C higher than the boiling!oint of the least e/a!orati/e com!onent in the sam!le- =ollo+ing these g'i,elines +ill hel! the'ser to obtain a GC s!ectr'm +ith goo, se!aration an, resol'tion-6"

Typical +ata of Expected ResultsGas chromatograms are !ro,'ce, +hen a sam!le is r'n on GC- 0he analytes in a sam!le

sho'l, be se!arate, base, on !olarity- =ig're @ sho+s a GC s!ectr'm of a methyl estermi&t're !asse, thro'gh a ca!illary col'mn-

31



2igure "! 'as chromatogram of a methyl ester mixture%

Results from the Class Experiment A mi&t're of ,ibromo!ro!ane an, ,ibromohe&ane +as r'n on a Shima,%' GC6"## instr'ment-0he tem!erat're of the inector !ort +as "###C. an, the col'mn tem!erat're +as "5##C- AHeli'm mobile !hase at a flo+ rate of @# m9min +as 'se,. along +ith a s!lit inection set'!- ANB65 col'mn. +ith a length of 5 mm an, an inner ,iameter of #-"5mm. +as 'se, for these!aration in this e&!eriment- n the GC chromatogra!h. ,ibromohe&ane has the longerretention time of the t+o molec'les- 0he chromatogram obtaine, in this e&!eriment ,is!layst+o !ea1s. +ith the ,ibromo!ro!ane on the left an, the ,ibromohe&ane on the right- 0he gas

chromotogram res'lts from the class e&!eriment are sho+n in =ig're 4-

32



Electron 0aramagnetic Resonance (E0R Spectroscopy

9asic 0rinciples: Electron !aramagnetic resonance s!ectrosco!y (EP<) also referre, to as electron s!in

resonance s!ectrosco!y (ES<) is a techni*'e 'se, to ,etect an, monitor ra,ical ions-

$nstrument 'rinciples:0he EP< instr'ment is com!ose, of a micro+a/e so'rce (lystron). an electromagnet.mo,'lation (Helmholt%) coils. an, a sam!le chamber- 0he mo,'lation coils /ary the magneticfiel,- S!ectra are recor,e, as first or secon, ,eri/ati/es- A schematic of an EP< instr'ment issho+n in =ig're -

2igure %! Schematic of E0R instrument! Reprinted fromlecture notes proided from I! Tanko!%

Method 'rinciples:EP< ,etects 'n!aire, electrons !resent in s!ecies +ith magnetically acti/e n'clei- EP< +or1sbase, on the ,e!en,ence of electron magnetic s!in states on the strength of an a!!lie,magnetic fiel,- 0here are t+o allo+e, *'ant'm s!in states. +hen there is no magnetic fiel,!resent these states are ,egenerate- An a!!lie, magnetic fiel, ca'ses the 'n!aire, electron toorient either !arallel or anti!arallel to the fiel, an, thereby se!arates the energy le/els- 0he

energy ,ifference bet+een the t+o states is ,escribe, by the follo+ing ∆E g β Ho +here g is a

constant. β is the Bohr magneton. an, Ho is the strength of the a!!lie, fiel,. 0his conce!t is

f'rther ill'strate, in =ig're "-

34



2igure .! +iagram sho=ing t=o distinct energy leels for the unpaired electron in thepresence of a magnetic field! Reprinted from lecture notes proided by I!Tanko!%

;hen the a!!lie, fiel, corres!on,s to the energy ,ifference. the system e&ists in a resonantstate- ;hen the energy matches the energy ,ifference. it is absorbe, by the sam!le an, a lineis !ro,'ce, on the s!ectra- 0he n'mber of lines obtaine, by a !artic'lar s!ecies is ,e!en,enton the s!in of the n'clei- 0his relationshi! is ,escribe, as "nI 3 . +here I is the s!in of then'clei an, n is the n'mber of e*'i/alent (by means of symmetry or ra!i, molec'lar motion)n'clei in the s!ecies- 0herefore for a s!ecies s'ch as methyl ra,ical one +o'l, e&!ect 4 linesthe three !rotons are e*'i/alent an, ha/e I 9". therefore "(@)(9") 3 4- 7'e to intrinsics!in ang'lar moment'm of some n'clei. a local magnetic fiel, is !resent an, can createa,,itional s!in states fora n'clei- 0his lea,s to hy!erfine co'!ling- Hy!erfine interactionsgenerally !ro,'ces line s!litting in the EP< s!ectra an, are ,escribe, by the hy!erfine co'!lingconstant. aH- ;hen there are more than one ty!e of magnetic n'clei !resent co'!ling occ'rsan, the n'mber of lines is ,etermine, by m'lti!lying the e&!ecte, n'mber of lines for each

acti/e n'clei- It sho'l, be note, that as a res'lt of instr'ment limitations an, co'!ling. not alllines are al+ays seen in the s!ectra- EP< ,etects the !resence of a ra,ical an, !ro/i,es someinformation abo't the local en/ironment of the ra,ical +hich can be 'se, to ,e/elo! str'ct'ralinformation- Sim'lation !rograms s'ch as ;inSim are 'sef'l for i,entifying s!ectra an,,eri/ing hy!erfine co'!ling constants- In or,er to ,etect short li/e, free ra,icals. EP< can becombine, +ith a techni*'e referre, to as s!in tra!!ing- In s!in tra!!ing the ra,ical of interest isreacte, +ith a nitroso or a nitrone com!o'n, to !ro,'ce a longer li/e, ra,ical that can be,etecte,-

9asic Sample Re@uirementsIn or,er to be ,etecte, +ith EP< the sam!le m'st contain a magnetically acti/e n'clei an, an'n!aire, electron- A,,itionally. the s!ecies m'st be long li/e, eno'gh to be ,etecte, +hile the

e&!eriment is r'n or a s!in tra! m'st be a!!lie,-

Typical +ata;Expected Results An EP< e&!eriment !ro/i,es a s!ectr'm ,escribing the local en/ironment of the 'n!aire,electron- 0he n'mber of lines obtaine, can be !re,icte, 'sing the e*'ation "nI 3 . +here I isthe s!in of the n'clei an, n is the n'mber of e*'i/alent (by means of symmetry or ra!i,molec'lar motion) n'clei in the s!ecies for s!ecies +ith more than one magnetically acti/en'clei the n'mber of lines e&!ecte, from each set of e*'i/alent n'clei are m'lti!lie, to

35



,etermine the total n'mber of lines in the s!ectra- 0he hy!erfine co'!ling constants can be,etermine, from the s!ectra by meas'ring the ,istances bet+een !ea1s-

Results from the Class ExperimentIn this e&!eriment EP< +as 'se, to analy%e a series of semi*'inone ra,icals generate, bybase in,'ce, o&i,ation of hy,ro*'inones- 0he follo+ing hy,ro*'inones +ere alay%e, .46

,ihy,ro&yben%ene (!6hy,ro*'inone). ."6,ihy,ro&yben%ene (o6hy,ro*'inone). "6methylhy,ro*inone. ".@6,imethylhy,ro*'ione. an, "6tb'tylhy,ro*'inone- 0he str'ct'res ofthese s!ecies are sho+n in =ig're @ an, generation of the ra,ical s!ecies is ill'strate, for p5hy,ro*'inone in =ig're 4-

2igure %&! Structures of analyzed species!

2igure %%! Reaction used to generate semi@uinone radicals! The reaction is demonstrated here=ith p1hydro@uione and occurs similarily (but at different rates for the other species!

0he hy,ro*'inones +ere ,issol/e, in methanol- J'st before analy%ing. the sam!les +erecombine, +ith H to in,'ce o&i,ation an, form the ra,ical s!ecies- !timal s!ectra +or1

36



recor,e, by /arying EP< !arameters incl',ing center fiel,. s+ee! +i,th. mo,'lation am!lit',e.!o+er atten'ation. an, gain- S!ectra +ere also sim'late, 'sing ;inSim- 0he e&!erimentallyac*'ire, an, sim'late, s!ectra are sho+n belo+ in =ig'res 56$. s!ectra for a o5hy,ro*'inonean, ".@6,imethylhy,ro*'inone +ere not s'ccessf'lly obtaine, in the e&!eriment- =or thesemi*'inone ra,ical generate, from p6hy,ro*'inone the fo'r !rotons on the ben%ene ring aree*'i/alent an, therefore fi/e lines are e&!ecte,. an, fi/e lines +ere obser/e, in the s!ectra as

seen in =ig're 5

Figure 12. Experimental and simulated EPR spectra for the

semiquione radical generated from p-hydroquinone. The hyperfine

coupling constant was set at !uass for the simulation.

=or "6methylhy,ro*'inone the three methyl !rotons are e*'i/alent an, +o'l, !ro,'ce 4 lines-0he three !rotons on the ben%ene ring are non e*'i/alent an, each s'ch that the total n'mber

of lines e&!ecte, is 4Z"Z"Z"@"- Clearly this n'mber of lines is not obser/e, in the s!ectrasho+n in =ig're F- 0he reason for this is !ea1 o/erla! ,'e to hy!erfine co'!ling-

37



Figure 1". Experimental and simulated EPR spectra for the semiquioneradical generated from 2-methylhydroquinone. The hyperfine coupling

constant was set at # !auss for all nuclei for the simulation.

0he semi*'inone ra,ical generate, from "6t6b'tylhy,ro*'inone has e*'i/alent !rotons +ithinthe t6b'tyl gro'! an, three non e*'i/alent !rotons on the ring- 0his in,icates that #Z"Z"Z"F#lines sho'l, theoretically be obser/e,- Again. the collecte, s!ectr'm ,oes not e&hibit theselines as a res'lt of o/erla! ,'e to co'!ling an, also !ossibly ,'e to instr'ment limitations- 0heco'!ling to the methyl gro'!s is e&!ecte, to be *'ite small-

Figure 1#. Experimental EPR spectra for the semiquione radical generated

from 2-t-$utylhydroquinone. The spectra was not accurately simulated.

S!ectra co'l, not be collecte, for o6hy,ro*'inone an, ".@6,imethylhy,ro*'inone ,es!itese/eral attem!ts- 0he s!ecr'm for o6hy,ro*'inone sho'l, ha/e sho+n lines from co'!ling to

the t+o e*'i/alent !rotons closest on carbon atoms @ an, F an, also to the t+o e*'i/alent!rotons on carbon atoms 4 an, 5- 0he s!ectr'm for the ra,ical obtaine, from ".@6,imethylhy,ro*'inone +o'l, be e&!ecte, to ha/e " lines the F methyl !rotons are e*'i/alentan, the t+o !rotons on the ring are e*'i/alent ($Z@")-

)pplications of the Techni@ue 6xidation and the Chemistry19iology #nterface:on%en et al4 'se, EP< to e/al'ate !otential antio&i,ant !ro!erties of /iolacein. a !igment!ro,'ce, by a bacteri'm that e&hibits !harmacological !ro!erties- EP< +as 'se, to,emonstrate that /iolacein can sca/enge hy,ro&yl ra,icals as in,icate, by a ,ecrease in the

38



hy,ro&yl ra,ical signal +hen /iolacein +as inc'bate, +ith ascorbate. iron chlori,e hy,rogen!ero&i,e an, 7EPMP as an a,,'ct- 0his along +ith the other analyses !erforme, len,scre,ence to the hy!othesis that /iolacein e&hibits in #i#o antio&i,ant !ro!erties-

2Malley et al also 'se, EP< in the conte&t of o&i,ati/e stress- 0hese researchers in/estigate,<S !ro,'ction from en,othelial cell mitochon,ria an, also e/al'ate, mito*inol. an antio&i,ant

that targets mitochon,ria-

Bertholon et al 'se," EP< to characteri%e ,r'g ,eli/ery nano!articles systems +ith!olysacchari,e com!onents grafte, onto the s'rface- hese researchers +ere able to a!!ly EP<to characteri%e the mobility of these nano!articles by labeling them +ith 46amino60EMP- 0heEP< s!ectra is altere, by Bro+nian motion of the label fast motion res'lts in +ell resol/e, lines+hile slo+ motion !ro,'ces broa,. e&ten,e, lines +ith interme,iary res!onses a!!earing ass'!er!ositioning of these line ty!es- 0he researchers ,emonstrate, that !olysacchari,e ty!ean, molec'lar +eight affecte, nano!articles mobility an, that fol,ing of the !olysacchari,e graft+as a critical factor-

S!in labeling of !olysacchari,es +as also 'se, by Steen et al.@ b't for a ,ifferent !'r!ose- 0he

interest of these researchers lies in characteri%ation of the sor!tion of !olysacchari,es tomineral se,iment s'rfaces an, the acti/ity of microbial e&tracell'lar en%ymes in this regar,. +itha!!lication to reminerali%ation !rocesses in the +ater col'mn- 0he researchers +ere able tosho+n by EP< that !olysacchari,e str'ct're an, se,iment ty!e affecte, s'rface loa,ing an,that EP< can be 'se, to obser/e en%ymatic hy,rolysis of !olysacchari,es-

References- Class notes Jim 0an1o. CHEM F5F4"- Bertholo. I- Hommel. H- abarre. 7- :a'thier. C- Pro!erties of Polysacchari,es Grafte, on

Dano!articles In/estigate, by EP<- 6angmuir! .&&/. "". 5485654#@- Steen. A-7- Amosti. C- Dess. - Blo'gh. D-:- Electron !aramagnetic resonance

s!ectrosco!y as a no/el a!!roach to meas're macromolec'le6s'rface interactions an,

acti/ities of e&tracell'lar en%ymes- Marine Chemistry . .&&/> # (@64). "FF6"$F-4- on%en. M- 7e Marco. 7- Cor,o/a. C-A-S- :ieira. 0-- Antonio. <-:- Crec%yns1i6Pasa.

0-B- Antio&i,ant !ro!erties of /iolcein !ossible relation on its biological f'nction-*ioorganic 7 Medicinal Chemistry . .&&/. 8@#$68@@-

39



#n1Situ 2T#R Spectroscopy

9asic 0rinciplesIn6sit' =0I< is a techni*'e that monitors reactions 'n,er real time con,itions- 0his techni*'e

!ro/i,es str'ct'ral an, 1inetic information abo't a reaction- 0he analysis is base, on the mi,6infra re, region. an, +hen the I< light e&cites the co/alently bo'n, atoms. it ca'ses the bon,sof a molec'le to /ibrate- Each f'nctional gro'! has a /ibration motion s!ecific for that gro'!+ithin the absor!tion fre*'encies in the I< s!ectr'm- 0h's *'alitati/e an, *'antitati/e ,ata canbe obtaine, for the ,ifferent f'nctional gro'!s +ithin a molec'le- Molec'les can ha/e /ibrationsthat can be symmetric or asymmetric. ben,ing. scissoring. t+isting. an, +agging motions. an,these motions are s!ecific for a !artic'lar +a/elength in the I< region- 6"

In this e&!eriment. the <eact I<X4### technology In6sit' =0I< S!ectrosco!ic e*'i!ment +as'se,- 0he 7iCom!0M ,iamon, A0< (atten'ate, total reflectance) !robe +as 'se, in ,irectcontact +ith the sam!le- 0he ,iamon, com!osite is inert an, chemically resistant- 0histechni*'e ,etects /ariations in reaction 1inetics. selecti/ity. an, reacti/ity in real time- It is non,estr'cti/e an, ,ata ac*'isition is contin'o's- 0he In6sit' =0I< ,etector is !'rge, +ith nitrogenan, bac1gro'n, an, calibration of a test scan are !erforme,- 0he alignment of the !robe iscritical for the !erformance of the test- Pea1 height ,etermination an, intensity +ere ma&imi%e,an, the !robe aligne, an, cleane,- ;hen the e*'i!ment +as rea,y. the reaction +asmonitore, 'sing a se*'ence r'n of "8 scans (e9a abo't " min'tes) for @# min'tes-6@

2igure %! #n1situ 2T#R Spectroscopy Tool!(http:;;glo!mt!com;mt;products;products;React#RH*&&&H0roduct0roductH%%&8*B".B",/7!Gsp

9asic Sample Re@uirementsIn6sit' =0I< can be 'se, for ,ifferent ty!es of sam!les as long as the !robe comes in ,irectcontact +ith the sam!le- In this e&!eriment. a li*'i, sam!le +as 'se,- ;e monitore, changesin the s!ectr'm ,'ring the Michael a,,ition reaction of ethyl acetoacetate (Et6AcAc) an, "6ehtylhe&yl acrylate (EHA)- 0he reaction +as cataly%e, 'sing.86,ia%abicyclo6L5-4-#6'n,ec6$6

ene (7B)- 0+o e&!eriments +ere con,'cte, one reaction +as !erforme, neat. an, the otherreaction +ith the a,,ition of the sol/ent tetrahy,rof'ran (0H=)-"

0he e&!erimental !roce,'re consiste, of 'sing a mol ratio of Et6AcAc9EHA. 'sing @ molO7B as the base catalyst- =or e&!eriment ^. a @6nec1 ro'n,6bottome, flas1 containing a stirbar +as charge, +ith g Et6AcAc- "-$44 g of EHA +as !'t in a /ial- 0he ro'n,6bottome,flas1 an, /ial containing EHA +ere !lace, in an oil bath V @5 UC- 0he 7B +as a,,e, an,allo+e, to calibrate for 5 min'tes- nce the first scan +as com!lete,. the EHA +as a,,e,.an, the scans +ere monitore, for changes in the s!ectr'm for @# min'tes- =or e&!eriment ^".

40



the abo/e !roce,'re +as follo+e, +ith the a,,ition of "-$44 g 0H= +hen the EHA +as a,,e,-In general. for the si%e of the flas1 +e 'se,. abo't @ ml of sam!le are nee,e, for the !robe tobe s'bmerge, +ithin the sam!le-"

Typical +ata or Expected Results and Results from the Class Experiment

2igure %! 2ull Spectrum of the Neat Reaction of ethyl acetoacetateand .1ethylhexyl acrylate!

2igure .! 2ull Spectrum of the Reaction of ethyl acetoacetateand .1ethylhexyl acrylate =ith tetrahydrofuran (TA2 Solent!

41



2igure "! ?aterfall 0lot of the Neat Reaction of ethyl acetoacetate and .1ethylhexyl acrylate!Sho=s fast decrease of C1C double bond J 8%& cm1%!

2igure *! ?aterfall 0lot of the TA2 Reaction of ethyl acetoacetate and .1ethylhexyl acrylate!Sho=s slo= decrease of C1C double bond J 8%& cm1%!

;e monitore, the ,ecom!osition of CHCH" in /inyl ethers in the EHA reactant o/er time- 0herange an, intensity for the carbon6carbon ,o'ble bon, is 8#685 cm6 (han,o't of f'nctionalgro'!s can classes of com!o'n,s +ith their absor!tion fre*'encies in the infrare, region.!ro/i,e, by Sharlene ;illiams)- 0h's. +e monitore, the ,ecom!osition of the ,o'ble bon, at8# cm6 for @# min'tes- 0he res'lts in,icate that a,,ition of 0H= ,il'tes the reaction. an, the,ecom!osition of the /inyl ether bon, is slo+er than the neat reaction- 0he ,o'ble bon,,ecreases +ith time to form a single bon,- )pplication of the Techni@ue to 6xidation and the Chemistry19iology #nterface

42



Beca'se =0I< is base, on the absor!tion of I< light by /ibrational transitions on co/alentbon,s. the intensities of s'ch /ibrations can gi/e *'antitati/e meas'rements of these bon,s-0h's. =0I< can be 'se, for a /ariety of a!!lications s'ch as i,entifying chemical str'ct'res. an,the e&tent of o&i,ation in biological materials-4 =or e&am!le. =0I< s!ectrosco!y can be 'se, to,etermine molec'lar str'ct'res of !roteins. n'cleic aci, m'tations. an, !ero&i,ation of

!hos!holi!i,s- 4 Selari1 et al-5 meas're, *'alitati/e changes in molec'lar str'ct'res in !oly(/inyl alcohol) (P:A) an, lactic aci, (A) com!o'n,e, !olymeric films an, +ere able to monitorthe esterification reaction bet+een P:A an, A film blen,s-

<eferences- 7r- ong In6sit' =0I< s!ectrosco!y class notes"- In6sit' I< laboratory han,o't@- Mettler60ole,o home!age

htt!99glo-mt-com9mt9!ro,'cts9!ro,'cts9<eactI<Q4###QPro,'ctPro,'ctQ#84@"@$F5-s!4- Petibois. C 7eleris. G- Chemical Ma!!ing of 0'mor Progression by =06I< 0o+ar,s

Molec'lar Histo!athology- rends *iotech. .&&/. "4. 45564F"-5- Se,lari1. : Saha. D 'rit1a. I Saha. P- Pre!aration an, Characteri%ation of Poly (/inyl

alcohol)9actic Aci, Com!o'n,e, Polymeric =ilms- $nternational J 'olym. Anal. Charact..&&/> . "5@6"$#-

43



)nalytical Sample 0reparation 3 -easuring c'04 5sing the 6xyScanSystem

9asic 0rinciples:

0his techni*'e relies on s!ectro!hotometric analysis of bloo, sam!les- A ,ecrease inabsorbance ,'e to o&i,ation of DA7PH in the sam!le is meas're, an, relate, to GP& acti/ity-

A,,itionaly. absorbance meas'rements are 'se, to *'antify the amo'nt of !rotein in the sam!lein or,er to con/ert the GP& acti/ity to a!!ro!riate 'nits- 0he ,etails of these analyses are,isc'sse, f'rther in the follo+ing-

$nstrument 'rinciples:Prior to loa,ing into the &yScan system re, bloo, cell sam!les ha, to be combine, +ith theb'ffer s'!!lie, as !art of the RIS cGPR assay 1it- Prior to loa,ing the +hite bloo, cellsam!les they ha, to be centrif'ge, to !reci!itate the membranes-

nce !re!are,. sam!les are loa,e, into the &yScan system by !i!etting 5#µ of sam!le into

the carosel !ortions beginning +ith !osition @ (!ositions an, " are fille, +ith "##µ of ,istille,

+ater to ser/e as o!tical blan1s)- 0he caro'sel is then loa,e, into the &yScan- 0heinstr'ment is !re!are, by lo+ering the reagent arm. closing the system an, in!'tting sam!leinformation- 0he assay is r'n thro'gh selecting the a!!ro!riate selectons on the ASSA men'-;hen the assay r'ns the instr'ment a,,s the sam!le to a sol'tion containing gl'tathione.gl'tathione re,'ctase. an, DA7PH- S'bse*'ently tert6b'tyl hy,ro!ero&i,e. the s'bstrate. isa,,e, to intiate the en%yme reaction- 0he absorbance at @4# nm is recor,e,- As DA7PSo&i,i%es to DA7P3 the absorbance at @4#nm ,ecreases in a manner !oro!ortional to the GP&acti/ity-

Method 'rinciples:0he metho, relies on ,etection of a ,ecrease in the absorbance at @4#nm +hich occ'rs +henDA7PH is o&i,i%e, to DA7P3- 0his o&i,ation in,irectly in,icates the le/el of cell'lar gl'tathione

!ero&i,ase acti/ity- 0he reactions sho+n in =ig're ,emonstrate the relation shi! bet+eenDA7PH o&i,ation an, GP&-

Figure 1. !lutathione peroxidase cataly%es the reaction of glutathione with a peroxide

to form oxidi%ed gluathione &!''!(. Reduction of !''! $ac) to !'* $y glutathione

reductase &!R( occurs silmutaneoulsy with oxidation of +,P* to +,P.

0he ,ecrease in absorbance at @4#nm is !ro!ortional to the GP& acti/ity an, the &yScansystem !ro/i,es GP& acti/ity ,ata in 'nits of m9m- 0his /al'e m'st be ,i/i,e, by the !roteincorrection /al'e to yiel, GP& acti/ity in m !er gram of !rotein- 0he !rotein correction /al'e isobtaine, by !erforming a !rotein assay to ,etermine the amo'nt of !rotein !er /ol'me ofsam!le- 0he !rotein assay relies on the 'se of Bio6<a, !rotein ,ye- 0he ,ye is !i!ette, intothe +ell of a micro!late. stan,ar,s (Bo/ine Ser'm Alb'min) an, sam!le are a,,e, to the +ell.

44



an, more ,ye is !i!ette, on to! of the sam!les- After inc'bating at room tem!erat're themicro!late is rea, on a s!ectro!hotometer at 55 nm- 0he Bio6<a, ,ye relies on CoomassieBrilliant Bl'e G6"5# 7ye- ;hen the ,ye bin,s to !rotein in sol'tion its absorbance ma&im'mshifts from 4F5 nm to 55 nm- 0he Bo/ine Ser'm Alb'min stan,ar, is 'se, to generate acalibration c'r/e an, s'bse*'ently meas'ring the absorbance of the sam!les at 55 nm allo+scalc'lation of the amo'nt of !rotein in the sam!le sol'tions-

9asic Sample Re@uirementsPro!er sam!le !re!aration is critical in analy%ing - Collecte, bloo, sam!les m'st be !rocesse,as *'ic1ly as !ossible once ,ra+n- 0his initial !rocessing ste! har/ests re, bloo, cells (<BCs).+hite bloo, cells (;BCs) an, !lasma- 0hese fractions sho'l, then be sna! fro%en an, store,at 6$#WC 'ntil analy%e,- ;hen sam!les are rea,y to be analy%e, they m'st be com!letelytha+e, an, tha+ing sho'l, be ,one at room tem!erat're-

Analytical Sample 'reparation=or this e&!eriment. sam!les +ere !re!are, !re/io'sly b't in or,er to gain a better'n,erstan,ing of sam!le han,ling !roce,'res <BCs. ;BCs. an, !lasma +ere isolate, frombloo, sam!les !rior to con,'cting the cGPR meas'rements-

;hole bloo, sam!les +ere !re/io'sly collecte, an, store, in he!arin containing t'bes-0o accom!lish this "5#µ of +hole bloo, +ere transferre, to a micro6centrif'ge t'be an, these

sam!les +ere centrif'ge, to isolate the re, bloo, cells- After centrif'ging the to! layers +ereremo/e, from the micro t'bes an, the remaining re, bloo, cell !ellet +as sna! fro%en in a ,ryice9ethanol bath- - 0o isolate the !lasma an, ;BC the +hole bloo, t'bes +ere centrif'ge,-Centrif'ging se!arates the layers the re, bloo, cells resi,e on the bottom. the +hite bloo, cellsform the mi,,le layer (the b'ffy coat layer) an, the !lasma forms the to! layer- 0he !lasma +as!i!ette, from the t'bes an, sna! fro%en- 0he b'ffy coat layer +as then isolate, from the t'bean, combine, +ith lysis b'ffer- 0he lysis b'ffer remo/es any re, cells that may ha/e also beencollecte, ,'ring this ste!- 0he t'bes +ith the ;BCs an, the lysis b'ffer are then centrifig'e,an, the b'ffer is ,ecante,- =resh lysis b'ffer is a,,e, an, the ;BC !ellet is re6s's!en,e, inthe b'ffer- Han1s Balance, Salt Sol'tion +as then a,,e, to the t'be an, t'bes +ere

centrif'ge, again- 0'bes +ere inc'bate, then centrif'ge, again an, the +ashing ste! +asre!eate,- 0he b'ffer +as then remo/e, again an, resi,'al li*'i, +as remo/e, by in/erting thet'be- =inally a small amo'nt of Han1s Balance, Salt Sol'tion +as a,,e, an, mi&e, +ith the;BCs- 0he ;BC sol'tion +as transferre, to a micro6centrif'ge t'be an, sna! fro%en-

Typical +ata;Expected Results0he &yScan system !ro/i,es GP& acti/ity in 'nits of m9m- Sam!les are r'n in tri!licate an,therefore three /al'es an, the a/erage are re!orte, for each sam!le- 0he !rotein assay!ro/i,es a stan,ar, c'r/e s'ch as the one obtaine, in this e&!eriment 'sing Bo/ine Ser'm

Alb'min sho+n in =ig're "- 0he !rotein assay then calc'lates an, re!orts the !roteinconcentration in the analy%e, sam!les in 'nits of mg9m- 0he assay re!ort also incl',esstan,ar, ,e/iations an, correlation /al'es for the analy%e, sam!les- 0hese /al'es are

combine, to 'ltimately !ro,'ce the ,esire, res'lt. cGP& acti/ity in 'nits of m9mg !rotein-

45



Figure 1/. 'ample protein cali$ration cur0e o$tained from the io-Rad Protein,ssay using o0ine 'erum ,l$umin.

Results from the Class ExperimentIn this e&ercise le/els of cell'lar gl'tathione !ero&i,ase (cGPR) in horses +ith rec'rrent air+ayobstr'ction (<A) +ere meas're, 'sing the &yScan system +ith the aim of a,,ressing thefollo+ing hy!otheses

o <A6affecte, horses ha/e higher <BC an, ;BC cG!& acti/ities com!are, to normal

control horseso 0he a,ministration of neb'li%e, magnesi'm s'lfate re,'ces <BC an, ;BC cGP&

acti/ities in a ,ose res!onse fashion

o 0he a,ministration of neb'li%e, magnesi'm s'lfate !l's a corticosteroi, (Alb'terol)f'rther re,'ces <BC an, ;BC cGP& acti/ities-

A,,itionally. !ro!er analytical sam!le han,ling +as ,isc'sse, an, e&!erience,- Si& treatmentmetho,s for the <A inflicte, horses +ere consi,ere, treatment Alb'terol sol'tion (!ositi/econtrol). " saline sol'tion (negati/e control). @ 6 lo+ Mg. 4 mi,,le Mg. 5 high Mg. F mi,,le Mg 3 Alb'terol- 0he obtaine, /al'es +ere com!are, to cGP& le/els of normal. healthyhorses +hich +ere meas're, !re/io'sly- 0he obtaine, /al'es for the control horses are sho+nin 0able - 0he treatment ,esign +as a F6treatment crosso/er ,esign an, treatments +erere!licate, o/er a " +ee1 !erio,- 0he <A horses +ere e&hibiting clinical <A as in,'ce, by!lacement in a nat'ral challenge en/ironment +hen treatments +ere a!!lie, an, sam!les +erecollecte,- 0he cGP& acti/ities for these horses are sho+n in 0able "-

Ta$le #. ellular !Px acti0ity le0els for control horses. 3alues were pre0iously measured and pro0ided to the

experimenters.

Aorse Name R9C c'0x (m5;mg protein ?9C c'0x (m5;mg protein

S'gar @#-"@ ""-@F

@5-F "4-#5

San,y @@-5@ "5-@

Ellie "5-F "5-F4

46



Penny @#-# "-45

Noe @"-F "F-#5

Table 7! Cellular '0x actiity leels for R)6 horses! R9C samples =ere done in duplicate =hile?9C cells =ere only analyzed once! The first horse listed in each treatment =as treated in thefirst collection period and the second horse listed =as treated in the second collection period> in

accordance =ith the crossoer design! The alues for Lips treatment . =ere preiously obtained!

TreatmentAorseName

R9C c'0x pre1treatment(m5;mgprotein

R9C c'0x post1treatment (m5;mg

protein

?9C c'0xpre1

treatment(m5;mgprotein

?9C c'0xpost1

treatment(m5;mgprotein

Co,y 4"F-5 ± @4-5$ 4"5-F@ ± 5#-" "#-$ @@-4

<'sty @$-$± F-F4 @"@-8# ± 5$-FF @@-## "-8

"Saran @8"-54 ± $"-#@ @F-"F ± #-4 44-@# 554-$F

i!s 5#-"5 F"-5 5@-$8 5#-F@

@<'sty @"F-8" ± #-@ @$5-8 ± -88 "@-45 "4F-@8

Baroness @@F- ± F-8@ @5-55 ± 4-8 @5-" "8-F

4i!s "85-$4 ± 4@-#@ "#-5 ± F-55 "#-44 ""@-5F

Co,y 44#-8F ± @-#" 44"-F ± @5-" 4F"-"# 44-4F

5Baroness @#-#$ ± $-#4 @5#-4" ± 5-#F "8-5# @#@-8@

Im! 4@F-F4 ± ""-"# 44F-@5 ± @5-55 "FF-"4 "8-$@

FIm! 4@-$ ± -F 48-4@ ± "-"# "5F-55 "8-8@

Saran 4@#- ± "-8" 455-# ± @-4" 4F5-F# @@4-"F

0he /al'es in 0ables an, " in,icate that horses +ith <A ,o in,ee, e&hibit higher cell'largl'tathione !ero&i,ase le/els than normal. healthy horses- 0o better /is'ali%e the effect of the

treatments bar gra!hs +ere generate, com!aring !re an, !ost treatment cGP& le/els- 0hesegra!hs are sho+n in =ig'res " an, @-

47



Figure 14. 'ummary of pre and post treatment R c !Px acti0ity le0els. The graphed

0aues are a0erages of the 0alues from $oth collection periods. Error $ars are standard

de0iations of these 0alues.

Figure /. 'ummary of pre and post treatment 5 c !Px acti0ity le0els. The graphed

0alues are a0erages of the 0alues from $oth collection periods. Error $ars are standardde0iations of these 0alues.

The seond h!"othesis an #e e$a%&ated #! o'"aring the effets of treat'ents 3 4 and 5 on

*x %e$e%s as these +ere the %o+ 'edi&' and high ,g treat'ents- .t a""ears fro' the data

that the highest dose of ,g (treat'ent 5) +as a#%e to red&e *x %e$e%s s%ight%! and thatthe 'idd%e dose of ,g (treat'ent 4) +as a#%e to red&e *x %e$e%s s%ight%! ho+e$er the

error in the 'eas&r'ents is signifiant The sa'"%es s&ggest that the third h!"othesis that

48



,g and %#&tero% red&es *x ati$it! the 'ost 'a! #e tr&e for s #&t the data does

not s&""ort this h!"othesis-

)pplications of the Techni@ue to 6xidation and the Chemistry19iology #nterface

7o'1i et al 'se, the gl'tathione !eroi&i,ase assay in their st',y in/estigating the biologicaleffects of e&!os're to ca,mi'm an, a static magnetic fiel,- 0hese researchers fo'n, thate&!os're res'lte, in increase, o&i,ation as in,icate, by M7A an, 86o&o,G'o. b't ,ecrease,antio&i,ant en%yme acti/ity- 0he a'thors !oint o't that bloo, sam!les +ere isolate, imee,iatelyafter collection an, that ali*'ots +ere fro%en an, store, at 68#WC.

Change et al 'se, the GP& assay in con'nction +ith other i,icators of o&i,ati/e stress to st',ythe effect of /itamin E an, e&ercise training on o&i,ati/e stress an, fo'n, that there is aninteraction bet+een /itamin E an, e&ercise training that effects GP& acti/ity-

References- Bio<a, Protein Assay 0ech Sheet

htt!99+++-fhcrc-org9science9labs9hahn9metho,s9biochemQmeth9biora,Qassay-!,f

"- Amara. S- Ab,elmele1. H Garrel. C- G'ira',. P- 7o'1i. 0- <a/anat. J6- =a/ier. A- Sa1ly.M- <ho'ma. -B- Infl'ence of static magnetic fiel, on ca,mi'm to&icity st',y of o&i,ati/estress an, 7DA ,amage in rat tiss'es- Journal of race Elements in Medicine and *iology ..&&/. "#(4). "F@6"F-

@- Chang. C6- H'ang. H6- 0seng. H6=- Hs''+. 67- 0so. 0-- Interaction of /itamin E an,e&ercise training on o&i,ati/e stress an, antio&i,ant en%yme acti/ities in rat s1eletalm'scles- he Journal of 3utritional *iochemistry . .&&,> 8(). @645-

49



6xygen Transmission Rate


&ygen transmission rate analysis (0<) is a techni*'e that is 'se, to meas're the amo'nt ofo&ygen that !ermeates thro'gh a !olymer film o/er a !erio, of time- 0y!ically. this techni*'e is'sef'l for analy%ing the efficacy of !ac1aging barrier films in !re/enting the o&i,ation of foo,.con,iments. ,rin1s. etc- 0he ,ata recor,e, for o&ygen !ermeation is re!orte, in 'nits ofcc9m"9,ay-6"

In o'r e&!eriment. an Illinois Instr'ments &ygen 0< mo,el 8## +as 'se, to analy%e,ifferent films- 0his s!ecific instr'ment is ca!able of tem!erat're an, h'mi,ity control. an, hasthe most e&!ansi/e meas'ring range a/ailable in the mar1et- Instr'ment o!eration first entailsattaching the sam!le to the ,iff'sion chamber- P're o&ygen is then intro,'ce, to the to!6half ofthe ,iff'sion chamber. +hile o&ygen6free gas is intro,'ce, to the bottom6half- &ygenmolec'les !ermeate thro'gh the film to the bottom6half of the ,iff'sion chamber. +here they are,etecte, +ith a co'lometric sensor-6@ An ill'stration of the ,iff'sion chamber is sho+n in fig're-

2igure %! Schematic of the 6TR diffusion chamber %

Method 'rinciples0he !rinci!les of this metho, are base, on the !ermeation !rocess. an, ho+ it is relate, tobarrier !olymer films- Permeation of films occ'rs in se/eral ste!s- =irst. the o&ygen molec'lescome into contact +ith the film- After contact occ'rs. the o&ygen molec'les m'st then a,sorban, ,issol/e into the !olymer film- 0he molec'les then ,iff'se ran,omly into the !olymer film.an, a net mo/ement of o&ygen molec'les across the film ens'es from the si,e in contact +iththe high concentration of o&ygen to the si,e in contact +ith the lo+ concentration- nce themolec'les mo/e across the barrier !olymer film. they ,esorb from the s'rface an, mo/e into theatmos!here- 0he !ermeability of a film. P. can be calc'late, by m'lti!lying the ,iff'sioncoefficient. 7. by the sol'bility coefficient. S-6@

0here are se/eral factors that affect !ermeability. incl',ing tem!erat're. h'mi,ity. an, a,,iti/es-Permeability increases +ith tem!erat're. es!ecially so if the tem!erat're is raise, abo/e the 0g of the !olymer film- H'mi,ity affects the !ermeability of certain !olymers- As h'mi,ityincreases. some !olymer films absorb +ater- An increase in +ater concentration can increase!ermeability for certain barrier !olymer films- =inally. +hen sol'ble a,,iti/es are blen,e, into a!olymer film. the ,iff'sion coefficient increases. +hich in t'rn increases the !ermeability of the

50



film- Ho+e/er. if an insol'ble a,,iti/e is blen,e, into the film. an increase or ,ecrease in!ermeability may be obser/e,-6@

9asic Sample Re@uirements0he only sti!'lation for a sam!le r'n on the 0< mo,el 8## is that the film thic1ness m'st beless than #-#5 inches- 0his instr'ment is s!ecifically ,esigne, for the 0< meas'rements of

!ac1aging barrier films- In a,,ition. this instr'ment is also ca!able of ma1ing 0<meas'rements on PE0 bottles-

Typical +ata or Expected Results0he instr'ment meas'res 0< as a f'nction of time- =ig're " sho+s an e&am!le of an 0<!lot obtaine, from a barrier !olymer film-

2igure .! Sample 6TR data obtained from #llinois #nstrument model 8&&%%

Results from the Class ExperimentSe/eral !olymer films +ere analy%e, 'sing 0< techni*'es- Polyami,e films of ,ifferentcom!ositions +ere characteri%e, along +ith a ,ry Mylar stan,ar, film- All sam!les +ere r'n at"@#C- 0able contains 0< ,ata for all of the films 'se, in the class e&!eriment- 0he 0<c'r/es for PAF6F an, the Mylar stan,ar, are sho+n in fig're @ an, 4. res!ecti/ely- 0he res'ltsobtaine, from the class e&!eriment of the t+o sam!le cells +ere not /ery re!ro,'cible. b't gi/ea general i,ea of the 0< of the films-

Table %! Results from class experiment

2ilm Cell ) 6TR (cc;m. ;day Cell 9 6TR (cc;m. ;day

PAF "5- @#-8

PAF6" - @-F

PAF6F $-$8 #-4

PAF68 F-8 #-5

Mylar Stan,ar, F-5 F5-8

51



2igure "! 6TR plot of 0)/1/

2igure *! 6TR plot of -ylar standard

)pplications of the Techni@ue to 6xidation and the Chemistry19iology #nterface0< a!!lications ty!ically in/ol/e barrier !olymers- 0+o e&am!les of recent 0< a!!licationsare as follo+s ) Jagannath et al- 'se, 0< to ,etermine the barrier !ro!erties of starch6caseinbase, e&tract films- 0hese meas'rements +ere !erforme, in con'nction +ith ,etermining the

52



antimicrobial !ro!erties of the films-4 ") Ja!anese researchers 'se, 0< to characteri%e thebarrier !ro!erties of silicone o&ynitri,e films for 'se in trans!arent foo, !ac1aging-5

References- >&ygen 0< Series 8###-? Illinois Instr'ments- "##$-

htt!99+++-illinoisinstr'ments-com9at8###-html-

"- >&ygen 0ransmission <ate-? ;i1i!e,ia- "##$-htt!99en-+i1i!e,ia-org9+i1i9&ygenQ0ransmissionQ<ate-

@- Bra,y. A-- Marsh. -S- 0he ;iley Encyclo!e,ia of Pac1aging 0echnology- "n, E,- De+ or1John ;iley an, Sons- $-

4- Jagannath. J- H- <a,hi1a. M- Dana!!a. C- M'rali. H- S- Ba+a. A- S- Journal of Applied'olymer Science .&&/. 040(F). @486@54-

5- I+amori. Sator' Gotoh. 'mi Moorthi. r%ys%tof- 8acuum. .&&.. 9- ("). @6$-

53



+issoled 6xygen -easurements

9asic 0rinciples=or the ,issol/e, o&ygen meas'rements +e 'se, the Mettler60ole,o !ortable o&ygen analy%er

in ta! 4###e- 0his e*'i!ment is base, on !olarogra!hic o&ygen meas'rements it is !ortable.an, meas'res o&ygen in sol'tion- 0he !olarogra!hic o&ygen sensor consists of a +or1ingelectro,e (catho,e). a reference electro,e (ano,e). an, an o&ygen6!ermeable membrane thatse!arates the electro,es from the sam!le- 0he transmitter s'!!lies a !olari%ation /oltage thatis a!!lie, to the catho,e- 0he o&ygen molec'les migrate thro'gh the !ermeable membranean, are re,'ce, at the catho,e- &i,ation occ'rs at the ano,e- 0he electrical c'rrent!ro,'ce, is !ro!ortional to the !artial !ress're of the o&ygen in the sam!le sol'tion-

9asic Sample Re@uirementsIn this laboratory e&ercise +e analy%e, an ascorbic aci, sol'tion (5 g9). commercial ##O!omegranate 'ice (,il'te, to !ro/i,e "$O 'ice). an, "$O cranberry 'ice-0he sam!le sol'tion of ascorbic aci, +as !lace, in a ro'n, bottom flas1 +ith " o!enings- 0heo&ygen meter !robe +as inserte, in one o!ening- 0he other o!ening +as seale, +ith a r'bbersto!!er- 0he sol'tion +as fille, all the +ay to the to! of the flas1. the flas1 +as in/erte, toremo/e o&ygen. an, more ascorbic aci, sol'tion inecte, thro'gh the r'bber sto!!er +ith asmall nee,le- 0he flas1 +as then !lace, in a +ater bath at @5 UC-

0he amo'nt of ,issol/e, o&ygen (mg9) in the ascorbic aci, sol'tion +as monitore, e/ery "min'tes for one ho'r (=ig're ")- 0he ,issol/e, o&ygen content of the !omegranate an,cranberry 'ices +as meas're, at one time only-

Typical +ata or Expected Results =ig're sho+s the ,issol/e, o&ygen content of #-5O ascorbic aci, sol'tion. "O cranberry 'icean, "O !omegranate 'ice o/er @ ho'rs (this ,ata +as r'n by John oont%)- 0he o&ygen,issol/e, in sol'tion ,ecrease, +ith time for all the sam!les- In general. the higher the amo'ntof antio&i,ants. the lo+er the ,issol/e, o&ygen content of the sol'tions-

54



2igure %! +issoled 6xygen Concentrations (mg;L of )scorbic )cid> Cranberry Iuice> and0omegranate Iuice )fter " Aours!

Results from the Class Experiment!In o'r e&!eriment. +e monitore, the ,issol/e, o&ygen content of 5O ascorbic aci, sol'tion forone ho'r- 0he collecte, ,ata is sho+n in =ig're "-

55



2igure .! +issoled 6xygen Concentration of 7K )scorbic )cid Solution )fter % Aour!

0his fig're sho+s that at 5O ascorbic aci,. the ,issol/e, o&ygen concentration ,ecreases o/ertime- Ascorbic aci, is a !o+erf'l +ater sol'ble antio&i,ant that sca/enges free ra,icals-

In a,,ition. +e meas're, the ,issol/e, o&ygen content of the 'ices at one !oint only- 0he "$Ocranberry 'ice +as F-@5 mg9 an, the "$O !omegranate 'ice +as #-F mg9- 0he cranberry

'ice ha, ##O /itamin C a,,e, +hereas the !omegranate 'ice ,i, not ha/e any /itamin Ca,,e,- 0herefore. the ,ifference in the ,issol/e, o&ygen content bet+een the 'ices co'l, beattrib'te, to the ascorbic aci, content-

)pplication of the Techni@ue to 6xidation and Chemistry19iology #nterface7issol/e, o&ygen meas'rements in a*'eo's systems are not only 'sef'l in meas'ring theeffects of antio&i,ants in the system as seen in this laboratory e&ercise- Benoit et al-" meas're,the ,issol/e, o&ygen content in the a'rentian Channel in Eastern Cana,a an, sho+e, theeffects of hy!o&ic +aters on marine life- 0his is significant since hy!o&ic o&ygen le/els in,icatefish s!ecies can not re!ro,'ce or s'r/i/e 'n,er limite, o&ygen con,itions- 0h's. ,issol/e,o&ygen meas'rements hel! biological ecosystems correct the !roblem of lac1 of o&ygen tos'stain life-"

0he !ortable ,issol/e, o&ygen analy%er can be 'se, to meas're the o&ygen content in thebe/erage in,'stry in !ro,'cts s'ch as beer. +ine. an, sot ,rin1s- 0he meas'rements of,issol/e, o&ygen hel! ,etermine !rocessing efficiency an, *'ality control g'i,elines- In thebre+ing in,'stry. many s'bstances containe, in beer are o&i,i%e, +hen the beer is e&!ose, toair an, these reactions occ'r /ery ra!i,ly- 0herefore. caref'l control of o&i,ati/e reactions,'ring heating. !aste'ri%ation or boiling are necessary to !re/ent off6fla/ors (bitterness). clarityof the beer (t'rbi,ity). an, o/erall shelf6life of the final !ro,'cts- In a,,ition. ,issol/e, o&ygen

56



meas'rements are 'se, in +aste +ater !lants an, to hel! ,etermine the effects of antio&i,antsin a*'eo's systems as seen in the laboratory among other 'ses-@

an et al-4 in/estigate, the effects of ,ifferent concentrations of ,issol/e, o&ygen on theo&i,ati/e stress of cells (*acillus s!- ="F) e&!ose, to mena,ione. an o&i,i%ing agent- 0hea'thors fo'n, that high ,issol/e, o&ygen concentrations (55O68#O) a'gmente, the o&i,ati/estress res!onse of the *acillus s!- cells +hen e&!ose, to mena,ione- 0he high o&ygen

concentration res'lts in formation of reacti/e o&ygen s!ecies (<S) ,'e to the transfer ofelectrons stim'late, by mena,ione ho+e/er. at lo+ ,issol/e, o&ygen concentrations. theformation of <S by mena,ione is stale, by the limite, s'!!ly of molec'lar o&ygen-4

References- aboratory Han,o't- Mettler60ole,o Portable &ygen Analy%er in 0a! 4##e-"- Benoit. P Gratton. M'cci. A- Mo,eling of 7issol/e, &ygen e/els in the Bottom +aters of

0he o+er St- a+rence Est'ary Co'!ling of Benthic an, Pelagic Processes- MarineChem. .&&/. #". @6@"-

@- htt!99+++-mt!ro-com Mettler60ole,o +ebsite- 7issol/e, &ygen in the Bre+ing Process-In0a!_ 4###e an, In0a!_ 4##4e Portable 7issol/e, &ygen Analy%ers- Portable 7

Analy%er-

4- 0an. G H'a. N i'. 7 7'. G Chen. J- Infl'ence of &ygen e/el on &i,ati/e Stress<es!onse of *acillus s!- ="F to mena,ione- 'roc. *iochem. .&&7. 4. $F46$F-

57



)nalytical Sample Extraction

9asic 0rinciple

Analytical 6ipid Etraction of Soy)eansi!i, from soybeans +as e&tracte, 'sing organic sol/ents accor,ing to the Bligh an, 7yere&traction metho, as mo,ifie, by Christie (")-

2igure %! 2lo= chart of soybean lipid extraction according to the 9ligh and +yermethod! Soybean sample =as made to contain 8&K =ater!

Determination of 6ipid ;idation in (oodsi!i, o&i,ation (a'too&i,ation) in fats an, oils occ'rs /ia a self !er!et'ating free ra,icalmechanism that res'lts in hy,ro!ero&i,e formation (!rimary !ro,'cts of li!i, o&i,ation)- 0hese!ro,'cts 'n,ergo scission to form al,ehy,es. 1etones. /olatile organic aci,s. an, hy,rocarbons(secon,ary or final !ro,'cts of li!i, o&i,ation)-"

In biological sam!les. incl',ing foo,s. o&i,ati/e reactions nee, to be caref'lly monitore,- Manymetho,s ha/e been ,e/elo!e, to meas're ,ifferent com!o'n,s that are forme, or ,egra,e,,'ring li!i, o&i,ation- It is 's'ally recommen,e, that " or more metho,s sho'l, be 'se, to'n,erstan, the !rogression of li!i, o&i,ation- Metho,s to ,etermine li!i, o&i,ation incl',e!ero&i,e /al'e. p6anisi,ine /al'e. /olatile organic com!o'n,s (i-e- he&anal). an, thiobarbit'ric

aci, (0BA) test-"

'eroide 8alue 5 'rinciple:Pero&i,e /al'e is ,efine, as the millie*'i/alents (mE*) of !ero&i,e !er 1ilogram of sam!le- 0hefat or oil sam!le is ,issol/e, in acetic aci,6chloroform sol'tion- A,,ition of e&cess !otassi'mio,i,e reacts +ith hy,ro!ero&i,es in the sam!le an, io,ine is liberate,- 0he sol'tion is titrate,+ith stan,ar,i%e, so,i'm thios'lfate 'sing a starch in,icator (E*'ation ). an, !ero&i,e /al'ecalc'late,-"

58

heat



<H 3 3 I6 ` <H 3 3 H6 3 I"

I" 3 starch (bl'e) 3"Da"S"@ ` "DaI 3 starch (colorless) 3 Da"S4F

Anisidine 8alue 5 'rinciple:

0he p6anisi,ine /al'e estimates the amo'nt of 6 an, 6 'nsat'rate, al,ehy,es ("6al1enals an,". 46,ienals). +hich are the secon,ary !ro,'cts of o&i,ation- 0he al,ehy,es react +ith theanisi,ine reagent to form a chromagen that is meas're, s!ectro!hotometrially- A toto& /al'e(total o&i,ation) can be 'se, to in,icate total o&i,ation of a sam!le 'sing both. !ero&i,e /al'ean, anisi,ine /al'e 00RA: 3 "P:-"

In this laboratory e&ercise. !ean't. soybean. an, canola oils +ere analy%e, for o&i,ation 'sing!ero&i,e /al'e (P:) to meas're hy,ro!ero&i,e formation @ an, anisi,ine /al'e (A:) to meas'real,ehy,es-4

0he oil sam!les +ere hel, for 8 ,ays at "5 UC 'n,er fl'orescent light in o!en containers that+ere reseale, (treate, sam!les)- Matche, sam!les +ere store, 'n,er similar con,itions as

state, abo/e. b't the containers +ere +ra!!e, in al'min'm foil an, the hea,s!ace +as fl'she,+ith nitrogen (control sam!les)-5

9asic Sample Re@uirements=or the li!i, e&traction. 5# g of soybeans +ere mi&e, +ith +ater to obtain 8#O moist're an, theBligh an, 7yer metho, follo+e, (=ig're )- =or the li!i, o&i,ation meas'rements. the officialanalytical metho,s for P: an, A: +ere follo+e,-

'eroide 8alue: An oil sam!le of 5-# #-#5 g +as +eighe, into a "5# ml Erlenmeyer flas1- 0hirty ml @ acetic

aci,6methanol +as a,,e, to ,issol/e the oil- ne half ml of sat'rate, I +as a,,e, an, letstan, for min- 0hen. @# ml of +ater +as a,,e, to sto! the reaction an, #-5 ml of starchin,icator sol'tion +as a,,e,- 0he mi&t're +as titrate, +ith either #-# D or #- D so,i'mthios'lfate 'ntil the yello+ color ,isa!!ears. an, a clear sol'tion is obtaine,- @

Anisidine 8alue: An oil sam!le of #-564-# g +as +eighe, in a "5 ml /ol'metric flas1 an, bro'ght '! to /ol'me+ith isooctane- 0he absorbance of the fat sol'tion in isooctane (Ab) +as meas're, against ablan1 of isooctane at @5# nm in a *'art% cm c'/ette- =i/e ml of the fat sol'tion +as !i!ette,into a # ml test t'be (test t'be A) an, 5 ml of isooctane into another t'be (test t'be B)- ne mlof anisi,ine reagent +as a,,e, to test t'be A an, B- 0he t'bes +ere /orte&e, an, left in the,ar1 for # min'tes- 0he absorbance of the fat sol'tion an, anisi,ine (As) +as meas're,

against the absorbance of the isooctane an, anisi,ine sol'tion at @5# nm-4

Typical +ata and Results from Class Experiment

Table %! 0eroxide $alue +etermination!

Treatment Sample =t!(g

mls &!&% NNa.S.6"

mls &!% NNa.S.6"

me@ peroxide;<gsample

Pean't oil 6control

5-4 -# @-$#

59



Pean't oil 6treate,

5-#5 -"5 "4-$

Soybean oil 6control

5-"# #-F# -5

Soybean oil 6treate,

5-#F #-4# $-#

Canola oil 6control

5-#F #-#5 #-#

Canola oil 6treate,

5-5 "-## @-88

Z Pero&i,e /al'e (millie*'i/alents !ero&i,e9g sam!le) S & D & ###9+t of sam!le (g)-;here S ml Da"S"@- D normality of Da"S"@-

Table .! )nisidine $alue +etermination!

Treatment Sample=t! (g

)bsorbance of 2atSolution ()b

)bsorbance of 2at Solutionafter )nisidine Reaction ()s

)$

Pean't oil 6

control

"- #-8 - 5-$"

Pean't oil 6treate,

"-# #-5# -#F# -5@

Soybean oil 6control

"-# #-"# -8@ 5-"

Soybean oil 6treate,

"-# #-"4$ #-85 -F

Canola oil 6control

4- #-"F 6 6

Canola oil 6treate,

4-# #"4 6 6

ZA: "5 & (-"As 6 Ab)9+t of sam!le (g)- 0he P: res'lts in,icate the treate, sam!les +ere more o&i,i%e, (higher le/els ofhy,ro!ero&i,es) com!are, to the control sam!les (0able )- 0he canola oil sam!les ha, lo+ P:for both treatments !robably beca'se an antio&i,ant ('s'ally /itamin E) is a,,e, tocommercially a/ailable canola oil to !re/ent li!i, o&i,ation ,'e to the high ,egree of'nsat'ration- Accor,ing to literat're /al'es. a fresh. high *'ality oil sho'l, contain a P:#-Pero&i,e /al'es d "# s'ggest !oor *'ality of the oil- =or e&am!le. soybean oil +ith P: of 65.56#. an, d# in,icate lo+. me,i'm. an, high le/els of o&i,ation- "

'r A: res'lts sho+e, that the control oil sam!les ha, higher le/els of al,ehy,es. an, the A:/al'es for !ean't an, soybean oils +ere /ery high- 0he A: /al'es for canola oils +ere not,etermine, !ossibly ,'e to e&!erimental error (0able ")-0he A: is a meas're of al,ehy,e formation ("6al1enals) that occ'rs after hy,ro!ero&i,es ha/ebeen bro1en ,o+n an, secon,ary !ro,'cts of o&i,ation are forme,- In o'r e&!eriment. theres'lts s'ggest the control sam!les +ere more o&i,i%e, than the treate, sam!les. +hich is incontra,iction +ith P:- 0h's. I belie/e the res'lts of A: +ere not acc'rate. an, the P: sho+e,o&i,ation of the treate, sam!les as e&!ecte,- Accor,ing to literat're /al'es. fresh soybean oilsho'l, ha/e an A: "-#-"

60



It +o'l, ha/e been hel!f'l to meas're P: an, A: of the oil sam!les at baseline (time # ,ays)an, after storage (time 8 ,ays) 'n,er the s!ecifie, con,itions. to ma1e com!arison of theo&i,ati/e changes that occ'rre, +ith time-

)pplication of the Techni@ue to 6xidation and the Chemistry19iology #nterfacei!i, e&traction is essential to obtain li!i,s from com!le& foo, matrices an, biological sam!les-

:alencia et al-5 e&tracte, li!i, from sa'sages that +ere fortifie, +ith omega6@ fatty aci,s in or,er to e/al'ate the e&tent of li!i, o&i,ation in the foo, sam!les- 0he a'thors meas're, P:(hy,ro!ero&i,es) an, thiobarbit'ric aci, (0BA) reacti/e s!ecies (meas're of secon,ary !ro,'ctsof o&i,ation i-e- al,ehy,es. malon,ial,ehy,e)- 0he 0BA metho, meas'res al1enals +hich issimilar to A:-F et et al-$ ,etermine, P: to e/al'ate the effects of ,ifferent antio&i,ants an, ra!esee, oil in anem'lsion system (i-e- mil1 s'!!lemente, +ith fish oil) in the absence an, !resence ofantio&i,ants s'ch as /itamin E an, ascorbyl !almitate- 0h's. P: is an analytical techni*'e'se, to ,etermine e&tent of li!i, o&i,ation in biological9chemical sam!les in combination +ithother metho,s s'ch as A:. 0BA. /olatile com!o'n,s. an, sensory-

References- aboratory Han,o't of i!i, E&traction- Christie- Mo,ification of Bligh an, 7yer- Can. J.

*iochem. 'hysiol. %B7B. @$. -"- Pi1e. A- =at Characteri%ation- In =oo, Analysis. @r, E,ition. Dielsen. SS. E,- l'+er

Aca,emic9Pleni'm P'blishers De+ or1. "##@. !!-""$6"4F-@- aboratory Han,o't of Pero&i,e :al'e- AAC fficial Metho, F5-@@ Pero&i,e :al'e of ils

an, =ats- JA;AC. %B/7. 48.$5-4- aboratory Han,o't of !6Anisi,ine :al'e- ACS fficial Metho, C,686#-5- aboratory Han,o't of Analytical E&traction aboratory an, 7etermination of &i,ation of

=oo, ils-F :alencia. I Ansorena. 7 Astiasaran. I- D'tritional an, Sensory Pro!erties of 7ry =ermente,

Sa'sages Enriche, +ith n6@ P=As- Meat Science. .&&/. $". $"@6$@@-$- et. MB Jacobsen. C Pham. A Meyer. AS- Protection Against &i,ation of =ish6il6

Enriche,6Mil1 Em'lsions thro'gh A,,itions of <a!esee, il or Antio&i,ants- J. Agric. (oodChem. .&&7. 5@. 54"654@$-

61



Sensory Ealuation

9asic 0rinciplesSensory !erce!tion factors in ,etermining the effects of o&i,ation in foo,s an, +ater systems

+ere e/al'ate, in this laboratory e&ercise- 0he h'man sensory !erce!tions in e/al'atingo&i,ation in foo,9+ater systems can be correlate, to analytical techni*'es to ,etermine thee&tent or !ercei/e, e&tent of o&i,ation in com!le& systems-

In this laboratory e&ercise. cons'mersT res!onses (affecti/e test 6 he,onic test). a ,irecte,assessment of an attrib'te of interest (,iscrimination test 6 ,irectional !aire, com!arison test).an, ,iscrimination of s'btle9small ,ifferences bet+een !ro,'cts (triangle test) +ere e/al'ate,by tasting !otato chi!s (affecti/e an, ,iscrimination tests) an, he&anal containing +atersam!les (triangle test) +ere e/al'ate, by aroma- In a,,ition. !re!aration of sam!les of thesensory techni*'es abo/e. +ere !resente, to /ol'nteers acting as !anelists-

Affecti#e Method: <edonic est 0his ty!e of test is !erforme, +hen information abo't ,egree of li1ing or ,isli1ing is ,esire,-0he ratings for each sam!le are gi/en n'merical /al'es ranging from li1e e&tremely () to ,isli1ee&tremely ()-"

Potato chi!s e&!ose, to /ario's treatments +ere teste, 'sing a !oint he,onic scale- 0hescale /arie, from ,isli1e e&tremely. ",isli1e /ery m'ch. @,isli1e mo,erately. 4,isli1eslightly. 5neither li1e nor ,isli1e. Fli1e slightly. $li1e mo,erately. 8li1e /ery m'ch. li1ee&tremely-

0he treatments. co,es. an, ran,omi%ation of the !resentation or,er are as follo+s

Pro,'ct Co,e Presentation r,er Chi!s A (fresh chi!s) @84 @84Chi!s B (chi!s store, in !lastic bags) 54" Chi!s C (chi!s store, in glass ars) 54"

Difference esting Method: Directional 'aired Comparison est In the !aire, com!arison test. a !air of co,e, sam!les is !resente, to the !anelists to com!area s!ecifie, characteristic. in this case o&i,i%e, fla/or- In this test only t+o sam!les arecom!are, at one time- 0he !robability of a !anelist selecting a sam!le by chance is 5#Otherefore. the statistical efficiency is not great- 0he !aire, com!arison test gi/es no in,ication of the ,egree of ,ifference bet+een the t+o sam!les. b't only ,etermines +hether there is a,etectable ,ifference or not-"

Potato chi! treatments +ere !aire,. the !resentation or,er +as ran,omi%e,. an, the !anelistas1e, to i,entify the sam!le that has the more o&i,i%e, fla/or bet+een the !airs-


Pair Sam!le9Co,e Sam!le9Co,e Presentation r,er B98F C9$$ BC" C9@@ A9FF" CA

62



@ A9"F B984 AB

;here A =resh chi!s. B Chi!s store, in !lastic bags. C Chi!s store, in glass ars-

Difference esting 5 riangle est

0he triangle test is a ty!e of ,ifference test in +hich a !anelist recei/es @ co,e, sam!les- 0+oof the sam!les are i,entical an, one is the o,, or ,ifferent sam!le- 0he !anelist is as1e, toi,entify the o,, sam!le- 0he res'lts are analy%e, base, on the !robability that if no ,etectable,ifference e&ists. the o,, sam!le +ill be selecte, by chance 9@ of the time-"

0his metho, is 'se, in *'ality control a!!lications to ens're sam!les are the same from,ifferent !ro,'ction r'ns- It is also 'se, +hen ingre,ient s'bstit'tion or changes inman'fact'ring !rocesses res'lt in ,etectable ,ifferences-=or this test. aroma of +ater containing he&anal +as e/al'ate,- He&anal is a !ro,'ct of li!i,o&i,ation. an, a grassy aroma is associate, +ith he&anal-

0he !anelist +as instr'cte, to smell the sam!les from left to right an, select the o,,9,ifferent

sam!le-


Set Sam!le9Co,e Sam!le9Co,e Sam!le9Co,e Presentation r,er A9""F A984$ B9@8 AAB" B9"" A9""" B9# BAB@ B9$$5 B9@" A9""8 BBA

;here AGrassy +ater. B7istille, +ater-

9asic Sample Re@uirements 0he !otato chi!s sam!les +ere store, 'n,er /ario's con,itions-) Potato chi!s +ere store, on glass ars for 8 ,ays 'n,er controlle, o&ygen atmos!here.nitrogen fl'she, b't e&!ose, to fl'orescent light-") Plastic bags +ere 'se, to e&!ose !otato chi!s to fl'orescent light an, o&ygen-@) Commercially a/ailable ayTs !otato chi!s !ac1e, in al'min'm layere, film +ere o!ene, 'st!rior to the tests-0hese sam!les +ere e/al'ate, 'sing he,onic test (li1eability test) an, ,irectional !aire,com!arison test (,iscrimination test)

;ater sam!les +ith an, +itho't he&anal +ere 'se, in aroma sensory e/al'ation 'sing a,ifference test (triangle test)- 0he concentration of he&anal 'se, +as 4# g9 (from PinarTs

st',y)-

Typical +ata or Expected Results and Results from Class ExperimentIn the first !art of the e&!eriment. +e !erforme, an, acte, as !anelists for all @ the sensorytests ,escribe, abo/eIn the secon, !art of the sensory lab. +e learne, the techni*'es of each test. an, !resente, thesam!les to /ol'nteer !anelists. an, the ,ata +as collecte,-

63



0he last !art of the sensory lab. ,escri!ti/e *'alitati/e metho, of coo1ies +as not !erforme,,'e to time limitations-

Results from Aedonic Test:

0able - <es'lts from He,onic Affecti/e 0est i1e, Potato Chi!s-

0anelist Name Chips ) Chips 9 Chips CS'san $ F

Annie @ 4+a1' $ 4e/in 8 @ 47r- 7'ncan 8 8 4im $ 4 "Chi!s Afresh Chi!s BPlastic bag Chi!s CGlass Jar-

Statistics <es'lts for ne ;ay AD:A of He,onic 0est 6 JMP gra!h

6ne=ay )nalysis of Aedonic 9y Chips

H e , o n i c

"

@

4

5

F

$

8

#

" @

Chi!s

All Pairs

0'1ey6ramer

#-#5

6ne=ay )noaSummary of 2it

<s*'are #-F$F A, <s*'are #-5FF"5<oot Mean S*'are Error -5F@4$"Mean of <es!onse 5-88888bser/ations (or S'm ;gts) 8)nalysis of $arianceSo'rce 7= S'm of S*'ares Mean S*'are = <atio Prob d =Chi!s " 5- "-555F "-## &!&&&,Error 5 @F-FFFFF$ "-4444

64



'r res'lts sho+e, that " !anelists fo'n, the !otato chi!s store, in glass ars to be the mosto&i,i%e, chi!s- Panelists chose the F !airs containing chi!s C e/ery time they +ere !resente,-0he !otato chi!s store, in the !lastic bags +ere the secon, most o&i,i%e,- Panelists chose!otato chi!s store, in !lastic bags to be more o&i,i%e, com!are, to the fresh chi!s- Done ofthe !anelists chose the fresh !otato chi!s (chi!s A) to be o&i,i%e,. as e&!ecte,-

0he !ac1aging of the !otato chi!s an, treatments ha, an im!act on the !ercei/e, ,egree ofo&i,ation- 0he fresh chi!s +ere commercial chi!s that are !ac1e, in al'min'm coate,m'ltilayer film an, nitrogen fl'she,- 0he !otato chi!s +ere seale, !rior to the lab- 0he !otatochi!s store, in the glass ars +ere nitrogen fl'she, an, e&!ose, to fl'orescent light an, +ere!ercei/e, more o&i,i%e, than the chi!s store, in the !lastic bags- Plastic is !ermeable too&ygen an, light ho+e/er. the chi!s in the ars may ha/e ha, a more ,irect contact +ith thelight res'lting in increase, o&i,ation-

oo1ing at 0able # for the n'mber of correct res!onses in ,irectional ,ifference test. for nF.(each !air +as !resente, F times. AB. AC. BC) the total n'mber of correct res!onses is F toachie/e a significant ,ifference at the 5O significance le/el- @ 0h's. chi!s C are the mosto&i,i%e, follo+e, by chi!s B an, chi!s A +ere fresh-

Results from +ifference Triangle Test:

0able @- <es'lts from 0riangle 0est Grassy ;ater-

0anelist Name 6rder of Samples()>9

Selected Sample Correct (M;N

+a1' AAB B S'san AAB A D

Annie AAB A DS'san BBA B D

+a1' BBA A Annie BBA A S'san ABA A D+a1' ABA B

Annie ABA A D

im AAB A Dim BBA A im BAB A 7r- 7'ncan AAB B 7r- 7'ncan BAB A 7r- 7'ncan BBA B D

e/in AAB B e/in BAB A e/in BBA A AGrassy +ater (4# g9 he&anal in ,istille, +ater) B7istille, +ater-

In this e&!eriment. o't of 8 !anelists +ere able to ,etect a ,ifference bet+een the grassy+ater an, the ,istille, +ater- oo1ing at 0able 8 for the n'mber of correct res!onses in atriangle test. for n8. # correct res!onses are nee,e, at the 5O le/el of significance to ,etecta ,ifference-@ 0h's !anelists o't of 8 in,icates that there is no statistically significant

66



,ifference bet+een the t+o sam!les at the #-#5 le/el- Panelists +ere 'nable to ,iscriminatebet+een the grassy +ater an, ,istille, +ater-

In my o!inion this test +as ,iffic'lt since the ,ifference +as /ery s'btle- I ha, to g'ess +hich+ater +as +hich-

)pplication of the Techni@ue to 6xidation and Chemistry19iology #nterfaceSensory e/al'ation is a !o+erf'l techni*'e to e/al'ate the e&tent of o&i,ation in biological an,chemical sam!les- Do analytical techni*'e can mimic the h'man sensory res!onse therefore.sensory e/al'ation is 's'ally 'se, in combination +ith analytical techni*'es- Pa!achrista' etal-4 e/al'ate, off6fla/or ,e/elo!ment an, acce!tability of mil1 sam!les store, 'n,er ,ifferentcon,itions- Mil1 sam!les +ere store, in PE0 bottles +ith or +itho't : bloc1ers. !a!erboar,cartons an, e&!ose, to fl'orescent light- 0he a'thors meas're, off6fla/or ,e/elo!ment in themil1 sam!les an, relate, the ,ata to chemical an, microbiological changes- Base, on thesensory e/al'ation. the a'thors fo'n, that PE0 an, : bloc1ers !ro/i,e longer shelf6life than!a!erboar, carton mil1 sam!les-4 Similarly. arsen et al-5 e/al'ate, sensory changes in e&tr',e, oats store, 'n,er ,ifferent!ac1aging. light. an, tem!erat're con,itions- 0he sensory res'lts +ere correlate, +ith changes

in hea,s!ace o&ygen concentrations. light e&!os're. an, o&i,ati/e stability- 0h's sensorye/al'ation +as 'se, to monitor chemical changes in a biological system s'ch as oats-5

F- <eferences-- aboratory Han,o't 6 Sensory Analysis aboratory-"- armon,. E- 6a)oratory Methods for Sensory E#aluation of (ood! Cana,a 7e!artment of

Agric'lt're P'blication F@$ tta+a Cana,a. 8" !! "#65-@- Meilgaar,. M Ci/ille. G: Carr. B0- Sensory E/al'ation 0echni*'es. @r, E,- C<C Press

;ashington 7-C-. !!- @F86@$- (han,o't !ro/i,e, in class)4- Pa!achristo'. C Ba,e1a. A Cho'liara. I on,yli. E o'rtis. ontominas. MG-

E/al'ation of Polyethylene 0ere!hthalate as a Pac1aging Material for Premi'm 'ality;hole Paste'ri%e, Mil1 in Greece- Eur. (ood. "es. echnol. .&&/. ""4. "@$6"4$-

5- arsen. H ea. P <o,botten. M- Sensory Changes in E&tr',e, at Store, n,er 7ifferentPac1aging. ight. an, 0em!erat're Con,itions- (ood. =ual. 'ref. .&&7. F. 5$@6584-

67



)nimal Cell Culturing

9asic 0rinciples:Cell c'lt're in/ol/es the gro+th of li/e cells o'tsi,e of a li/ing organism- Cells are remo/e,

from organ tiss'es !rior to or ,'ring c'lti/ation. an, are s'staine, in a laboratory setting- Cell!assaging or s'bc'lt'ring. !rolongs the time that a !artic'lar cell line can be c'lt're, by!ro/i,ing fresh n'trients an, room for cell gro+th to contin'e- 0here are three ty!es of cellc'lt'res !rimary cell c'lt'res. contin'o's cell c'lt'res. an, cell lines- Primary c'lt'res areobtaine, ,irectly from a tiss'e so'rce an, e&hibit the closest relation to act'ally in #i#o cells b'tare short li/e,- Contin'o's c'lt'res can be !ro!agate, in,efinitely 'sing chemical treatmentsor /iral oncogenes- 0hese c'lt'res ,o not retain in #i#o characteristics of the cells b't easy togro+ an, store. are longer li/e, than !rimary cell lines an, ha/e little batch /ariability- 0he cells+or1e, +ith in this e&ercise +ere a contino's c'lt're- Cell lines are a single cell ty!e that are!ro!agate, in c'lt're for a limite, n'mber of ,i/isions- Cell c'lt're systems can be eithera,herent cells. +hich are those gro+n as a monolayer on a glass or !lastic s'bstrate. an,S's!ension cells +hich are those that float free in c'lt're me,i'm- In this e&ercise +e +or1e,+ith a,herent cells-

A!!lications of cell c'lt're incl',e in/estigation of basic cell biology an, biochemistry.e/al'ation of the effects9interactions of ,r'gs. n'trients. ,isease ca'sing agents. an, othercom!o'n,s on9+ith cells. to&icity testing. cancer research. /irology. /accine !ro,'ction. tiss'eengineering. gene thera!y. an, genetic engineering-

$nstrument 'rinciples:Cell c'lt'ring re*'ires m'lti!le !ieces of e*'i!ment- A C" inc'bator is necessary to maintain!hysiological !arameters incl',ing a tem!erat're of @F6@$WC. 56#O C" le/el. !H $-"6$-5. an,a h'mi, en/ironment- A,,itionally. a laminar flo+ hoo, is re*'ire, to maintain sterile con,itions+hile +or1ing +ith the cells- Sterile laboratory glass+are2 incl',ing flas1s. !i!ettes an, c'lt're,ishes are necessary for transferring cells an, c'lt're me,ia. an, storing an, gro+ing cells- Inor,er to /is'ali%e the cells it is necessary to ha/e an in/erte, light microsco!e- If cells are to beco'nte, hemacytometer !lates are re*'ire,- Hemacytometer !lates +ith t+o etche, glasschambers that contain a s!ecific /ol'me of cell s's!ension- 0he chambers ha/e co'nting gri,sthat are /isible 'n,er the in/erte, light microsco!e an, allo+ one to co'nt the cells in a s!ecific/ol'me of cell s's!ension an, s'bse*'ently ,etermine the amo'nt of cells in the totals's!ension by m'lti!lying by the a!!ro!riate ,il'tion factor- It is also necessary for a cellc'lt're laboratory to ha/e cryo!reser/ation e*'i!ment. an, refrigerator an, free%er s!ace tomaintain cell stoc1s an, c'lt'ring s'!!lies-

Method 'rinciples: A,herent cells are s'bc'lt're, by first remo/ing ol, me,ia from the !re/io's !assage an,+ashing +ith a salt sol'tion s'ch as Han1s Balance, Salt Sol'tion (HBSS) or Phos!hate B'fferSol'tion (PBS)- 0he +ash ste! remo/es ol, me,ia an, nona,herent cells- 0ry!sin is thena,,e, to ,etach the cells from the c'lt're flas1- 0he res'lting s's!ension is then ,il'te, an,s's!en,e, in fresh c'lt're me,ia- 0he cells are then see,e, in a fresh c'lt're flas1 an, !lace,in the C" inc'bator- Cell gro+th is monitore, 'sing an in/erte, light microsco!e- A,herentcells a!!ear elongate, +hile ,etache, cells are ro'n, an, mo/e freely- It is ,esirable to ha/ealmost a f'll monolayer of a,herent cells (b't not more than a f'll monolayer) !rior to 'tili%ingc'lt're, cells for other analyses- Cell gro+th is ,escribe, by stating a !ercentage of co/erageof the flas1 bottom. a com!letely co/ere, c'lt're flas1 +o'l, be referre, to as >##O confl'ent-?

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Cells can also be co'nte, by !lacing a ,ro! of c'lt're on a hemacytometer an, co'nting 'n,erthe in/erte, light microsco!e-

9asic Sample Re@uirements<e*'irements for s'ccessf'l cell c'lt're +or1 incl',e re!lication of !hysiological con,itions (@F6@$WC. !H $-"6$-5. h'mi,ity. an, a!!ro!riate e*'ilibri'm bet+een biocarbonate concentrate an,

C" gas). an a!!ro!riate n'trient s'!!ly (,e!en,ent on cell ty!e). attainment of re*'ire,chemicals (balance, salt sol'tion. antibiotic. an, try!sin) an, sterile laboratory s'!!lies.en/ironment an, techni*'es- 0he me,ia m'st first be !re!are, an, sterili%e,- Cell gro+thme,ia consists of inorganic salts to reg'late memberane !otential an, maintain osmoticbalance. carbohy,rates to !ro/i,e energy. amino aci,s. /itamins. fatty aci,s an, li!i,s. !roteinsan, !e!ti,es. ser'm. an, antibiotics to !re/ent bacterial gro+th-

Typical +ata;Expected Results;hen s'bc'lt'ring cells one e&!ects to obser/e s'ccessf'l cell gro+th thro'gh the in/erte, lightmicrosco!e- Healthy. a,herent cells sho'l, a!!ear elongate,. in,icating that they are attache,to the gro+th s'rface- Imme,iately after !assage the cells sho'l, only co/er a small fraction ofthe flas1 or c'lt're ,ish b't if s'bc'lt'ring +as s'ccessf'l. the cells +ill gro+ to form a

monolayer- 0he time ta1en for the cells to co/er the s'rface ,e!en,s on the cell ty!e. gro+thme,ia. an, gro+th con,itions- ;hen cells form a monolayer they are referre, to as confl'ent or ##O confl'ent- If ,esire,. a cell co'nt can also be re!orte,- A!!ro!riate cell co'nt n'mbers,e!en, on the si%e of the flas1 or ,ish that the cells in +hich the cells are s'bc'lt're,-

Results from the Class ExperimentIn this e&!eriment h'man brain micro/asc'lar en,othelial cells +ere !assage,- Cells ha, been!re/io'sly !assage, by - ee- 0hese cells +ere obser/e, 'n,er the in/erte, light microsco!ethen !assage, 'sing <PMI F4# for HBMEC me,ia- 0he me,ia contains !enicillin6stre!tomycin. so,i'm !yr'/ate nonessential amino aci, blen,. /itamin mi&t're blen,. hea!artinen,othelia cell gro+th s'!!lement. fetal bo/in ser'm. D'Ser'm. an, basal me,ia <os+ell Par1Memorial Instit'te (<PMI) F4#- After being !assage, the cells +ere co'nte, 'sing a

hemacytometer- 0hirty eight cells +ere co'nte, in the hemacytometer gri,. corres!on,ing to@-8 & #F cells !er c'lt're flas1- 0he co'nts obtaine, by all class members are s'mmari%e, in0able - Cells +ere 5O confl'ent t+o ,ays after !assage- As the !'r!ose of this e&ercise +asto learn the techni*'es in/ol/e, in cell c'lt'ring the cells +ere not 'se, to obtain res'lts.ho+e/er the s'bc'lt're, cells co'l, ha/e been 'se, for st',ies s'ch as those mentione,!re/io'sly-

Table /! Cell Counts from passage of A9-EC cells! #deal cell count for the size flasks used (T,7is /1%& million cells per flask!

0assaged by: Cell Count (cells per culture flask

7r- - ee 8-# & #F

A- Aigster -F & #$

J- Goff "-5 & #F

S- Hannah @-8 & #F

S- Hogan @-5 & #F

- Hollan, -# & #F

B- Stane1 8-@ & #F

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G<cGP&SSG

A7P

H

GSH

final colab notebook.doc

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